In this study, the potential use of ancient plant ingredients in emerging bakery products based on possible prehistoric and/or ancient practices of grinding and breadmaking was explored. Various ancient grains, nuts and seeds  were ground using prehistoric grinding tool replicas. Barley-based sourdough prepared by multiple back-slopping steps was added to dough made from einkorn alone or mixed with the above ingredients (20% level) or commercial flours alone . Sieving analysis showed that 40% of the einkorn flour particles were >400 μm, whereas commercial barley and common wheat flours were finer. Differential scanning calorimetry revealed that lentil flour exhibited higher melting peak temperature and lower apparent enthalpy of starch gelatinization. Among all bread formulations tested, barley dough exhibited the highest elastic modulus and complex viscosity, as determined by dynamic rheometry; einkorn breads fortified with linseed and barley had the softest and hardest crust, respectively, as indicated by texture analysis; and common wheat gave the highest loaf-specific volume. Barley sourdough inclusion into einkorn dough did not affect the extent of starch retrogradation in the baked product. Generally, incorporation of ancient plant ingredients into contemporary bread formulations seems to be feasible. Recent archaeological research has revealed a wide range of plant materials, preserved mainly as charred plant remains, which have been most likely used for preparation of human food ,3,4,5,6.Triticum. monococcum, T. dicoccum and T. timopheevi, respectively). During the Bronze Age, spelt wheat (T. spelta) also appears for the first time at the end of the 3rd millennium B.C., while the free-threshing wheats  had a more limited occurrence in prehistoric times [T. aestivum and T. durum) have already been grown since the Neolithic period. These free-threshing cereals, over time, became the dominant cereals grown for food in Western Asia and Europe at the expense of the glume wheats, perhaps because the latter have a lower productivity per cultivated area, a higher bulk volume when stored in their glumes and a greater labour input required for dehusking before milling [Hordeum vulgare) grain and lentil (Lens culinaris) seeds, as well as seed of acorn nut (Quercus sp.), linseed (Linum usitatissimum) and poppy seed (Papaver somniferum) transformed into flour by grinding [The archaeobotanical record reveals that a wide range of cereals and pulses constituted the staple foods of prehistoric European communities ,8. In thic times . Nowaday milling ,11. An a milling . Apart fgrinding ,6.The transformation of cereals and other plant-derived food materials would have taken place with the facilities available at the time, which included stone grinding and pounding implements , cookingConsumer interest in ancient wheat species  with regard to their use in bakery products has recently emerged, particularly because these grains are rich sources of bioactive components and hence suitable for producing high value food products with enhanced nutrient content and health benefits, especially when used in the form of whole flours ,20,21. FIn this context, fortification of bakery items with ancient grains and seeds could have an impact to the wellbeing of individuals and contribute to the reduction of risk factors and/or management of chronic diseases, such as diabetes type 2 and cardiovascular diseases. Furthermore, adopting ancient breadmaking practices could give rise to new bakery products with improved nutritional attributes, health benefits and minimally processed food items, without additives and improvers, thereby satisfying the new consumer’s preferences and emerging life-trends related to ‘clean labelling’ of food products, healthy diets, and wellbeing.In the present study, experimental breads were prepared under controlled conditions using plant ingredients identified in the prehistoric record and resembling some of the prehistoric and/or ancient breadmaking practices  in an effort to establish in ongoing studies  a relevaT. monococcum), barley (Hordeum vulgare), acorn (Quercus sp.), lentil (Lens culinaris), poppy seed (Papaver somniferum) and linseed (Linum usitatissimum)—were ground using prehistoric grinding tool replicas constructed for the PLANTCULT project (T. aestivum), type T70 (white flour), and spelt wheat (T. spelta), whole flour, were also employed for breadmaking, whereas a commercial barley whole flour was also used for breadmaking and sourdough preparation. All the above three commercial flours were organic, provided by a local supplier .Five different plant raw materials—einkorn wheat , 5-mesh (0.80 mm), 6-mesh (0.65 mm), 8-mesh (0.50 mm), 10-mesh (0.40 mm), 16-mesh (0.24 mm), 20-mesh (0.20 mm), 30-mesh (0.13 mm), 40-mesh (0.10 mm) and 70-mesh (0.063 mm). For particle size analysis of einkorn flour, all of the above sieves were used, whereas for the commercial flours of barley and common wheat, only the last six sieves were employed. After shaking for 15 min by a mechanical vibratory shifter, the amount of flour retained on each sieve (WS) was weighed, and the contents of retained fractions were calculated as the percentage of the initial  flour weight as follows:The cumulative percent passing through the sieves was calculated by subtracting the cumulative percent of retained fractions from 100%.50 (median diameter) and d90 that are commonly used in the classification of ground materials were estimated, representing 50% and 90%, respectively, of the particles with diameters smaller than the specified values.The particle size parameters of dw/w solids were sealed hermetically into DSC aluminum pans. The pans were heated from 8 to 120 °C at a heating rate of 5 °C/min; samples were heated under a continuous flow of dry N2 gas (20 mL/min) to avoid moisture condensation during measurement. Three specimens from each flour preparation were analyzed by DSC. Parameters estimated from the DSC thermographs were the onset (To) and peak (Tm) starch gelatinization temperature, as well as the apparent starch gelatinization enthalpy (ΔΗ) calculated from the area of the endothermic (melting) peak, following calibration of the calorimeter with indium.The starch gelatinization properties of flours and flour mixtures  used forw/w) was used, following a recipe provided in a modern sourdough preparation book for the wider public [The sourdough used for breadmaking was prepared for project PLANTCULT by the traditional method of spontaneous back-slopped sourdough (Type I); all the preparation steps are presented in detail in r public . The user public . Followiw/v), followed by decimal dilutions in the same saline solution. Enumeration of microorganisms was carried out using the pour plate technique. Lactic acid bacteria were enumerated on De Man Rogosa and Sharpe agar (MRS agar)  containing natamycin, following incubation at 30 °C for 3 days, while for enumeration of yeasts, Yeast Malt Agar (YM agar) , containing tartaric acid was used after incubation at 25 °C for 5 days.For the determination of colony forming units (CFU/g) in mature mother sourdough and the final back-slopped barley-based sourdough used for breadmaking, 10 g portions of sourdough were homogenized with 90 mL NaCl  determinations, 10 g of sourdough were mixed with 90 mL of sterile distilled water, and the resultant suspension was kept under continuous stirring during both measurements. The pH was measured with a Bante 210 pH meter , and the TTA was determined by titration of the sourdough suspension with 0.1 N NaOH until a final pH of 8.4; the TTA values were expressed as mL of 0.1 N NaOH/g of sourdough.w/w), and followed by hand-kneading for 5 min. Before rheological testing, all dough samples were wrapped with a plastic membrane to avoid water loss and rested at room temperature for 20 min, allowing uniform moisture distribution and dough matrix relaxation before testing. The dough preparation procedure for each formulation was repeated in triplicate.For the study of dough rheological properties, flours obtained with the prehistoric grinding tools were passed through a coarse sieve (~1 mm) to remove all coarse particles of husk and bran and thus increase their homogeneity and improve their water retention capacity. Dough samples from all tested flours used for breadmaking  without Oscillatory measurements of doughs were performed by a rotational Physica MCR 300 rheometer  using a parallel plate geometry (50 mm diameter and 2 mm gap) with a solvent trap to avoid moisture loss during measurements; the plate had a sanded surface to prevent slippage of the measuring fixture. The temperature was regulated at 20 °C by a controlled temperature peltier system (TEZ 150P/MCR) with an accuracy of ±0.1 °C. After loading, the dough sample was left to further rest in the geometry for 15 min prior to measurement. Oscillatory measurements were performed, at which the storage (or elastic) modulus, G′; loss modulus, G″; and complex viscosity, η*, at a strain level of 0.01%, were monitored over an angular frequencies range of 0.1–50 Hz. The data of the rheological measurements were analyzed with the supporting rheometer software US200 V2.21.w/w on a flour basis (DY 200); i.e., the amount of flour from sourdough was 20g in 100g of total flour in the bread formulation. The level of added water in all dough formulations, including the sourdough water amount, was 80% (flour basis); the same water level was also added to the control sample.Breads were made from einkorn, spelt, barley and common wheat flours alone as well as from flour mixtures of einkorn with another grain or seed flour, i.e., spelt, acorn, lentil, barley, poppy seed and linseed (einkorn: other flour in an 80:20 ratio), with the addition of sourdough; all bread formulations are shown in For the breadmaking process, flours obtained with the prehistoric grinding tools were used without removal of the coarse particles of husk and bran in order to closely resemble the prehistoric practices. Actually, pieces of cereal bran and husk embedded in a cereal-based amorphous matrix (gelatinized starch) have been identified in several archaeological sites in Southeastern Europe, indicating that breadmaking and/or porridge making from cereal grains were possibly widespread food preparation methods in prehistoric times, by employing relatively simple processing tools and practices .The flow chart presented in Large deformation mechanical properties of the experimental breads were examined by a puncture test using a Texture Analyser  calibrated with a 5 kg load cell. The bread loaves were compressed with a spherical probe (0.635 cm diameter) up to rupture of the crust at a crosshead speed of 0.4 mm/s. Hardness of the crust was taken as the peak force of the force-displacement curve; two values obtained from the same loaf were averaged, and two loaf breads from each breadmaking process were averaged into one replicate.The volume of the bread loaves was determined with a homemade volume meter made from plexiglass and based on the rapeseed displacement method . Bread lSuspensions in ethanol of einkorn and commercial barley flours, barley sourdough, einkorn dough without and with sourdough, as well as crumb of fresh einkorn bread without and with sourdough were prepared under stirring. Aliquots of suspensions were taken by aspiration and examined using an Olympus BX51 microscope (Japan) equipped with dry lenses, a microscope digital camera Olympus DP70 and the Olympus micro DP70 software. The microscopic observation was carried out after staining of starch with iodine solution in bright field and cross polarized light in order to observe the starch granules and the changes in starch microstructure during dough preparation and breadmaking in the presence or absence of sourdough. At least ten captures for each sample were taken.w/w solids were hermetically sealed into DSC aluminum pans and heated from 8 to 120 °C at a heating rate of 5 °C·min−1. Three crumb specimens of each bread preparation from all the breadmaking processes were tested by DSC. The onset melting temperature (ToRET), the peak (TmRET) melting temperature and the apparent melting enthalpy (ΔHRET) of the retrograded starch were determined.Changes in starch physical state upon breadmaking and bread storage were also examined by DSC analysis of the crumb of fresh and staled einkorn bread without and with sourdough; for the staling events in the starch matrix, bread loaves were stored in sealed polypropylene bags at 3 °C for 6 days. Before analysis, crumb samples from fresh and stored bread were lyophilized and then ground into fine powder using liquid nitrogen. Aqueous slurries of the lyophilized samples (about 25–30 mg) containing 35% All physicochemical parameters of flours were tested in triplicates. Mean values of dough parameters, loaf specific volume and crumb retrogradation parameters were the average from three dough specimens, bread loaves and crumb samples, respectively (one from each dough or bread making procedures). For crust hardness, firstly, values from two different points of the same loaf were averaged, and the mean values presented in this study were obtained from two different breads that averaged into one replicate of the three breadmaking repetitions.Statistical analyses were performed by the IBM SPSS statistical software . All parameters of flour, dough and bread properties were analyzed by a one-way ANOVA, according to a generalized linear model, examining the effect of flours and their mixtures at all breadmaking stages. Differences between mean values were compared using the Tukey’s test at a = 0.05 significance level.50 and d90 values of einkorn, barley and common wheat flour were 287, 228, 123 μm and 879, 313, 252 μm, respectively. Thus, einkorn flour exhibited the largest particle size at all particle size distribution ranges among the three tested flours since einkorn grains were ground by prehistoric grinding tool replicas (stone grinding), in contrast with the other two commercial flours, which were finely ground by industrial mills. Additionally, between the two commercial flours, barley flour had larger particles as it was a whole flour compared with those from common wheat, which was a white flour (70% extraction rate); it is well-known that whole flours include bran and thus have higher mean particle size compared with flours originating mostly from the endosperm of cereal grains (white flours).Analysis of sieving classifies the flour particles by size as well by shape. The particles of flour are usually spherical-like, such that their diameters correspond to the sides of the square sieve opening. Particle size cumulative distribution curves of einkorn, barley and common wheat flour are presented in 50 = 350 μm) exhibiting increased stiffness and resistance to deformation and flow compared with that of a fine (d50 = 200 μm) barley flour preparation [According to previous studies, the particle size of flours significantly affects the rate of water absorption during dough making, as fine particles absorb water faster due to their greater surface area . Smallerparation . Howeverparation ; therefoparation ,22,23.w/w solids) of tested flour samples showed the typical endothermic peak of starch gelatinization at around 56.0–80.0 °C (o) of this endothermic transition and the disruption (melting) of starch molecular orders  upon gelatinization occurs at the peak temperature (Tm)  ; the areo (64.7 °C) and Tm (74.0 °C) and the lowest ΔH (0.9 mJ/mg) values among all tested flour samples, implying higher resistance of this legume starch towards gelatinization and a relatively lower amount of double helical structures (primarily involving the amylopectin component) compared with those of cereal starches , which have ~20–25% and 60–70%, respectively [o temperature (56.2 °C) followed by the common wheat (57.6 °C), while To values for flours of barley, einkorn and its mixture with lentil, spelt and barley displayed higher values, ranging from 58.9 to 59.4 °C . Moreover, barley flour showed significantly lower apparent enthalpy and higher onset temperature of gelatinization compared with common wheat flour , while the number of yeasts is limited . In our p < 0.05) higher elastic modulus and complex viscosity values compared with all doughs made from any wheat species flour , suggesting that the barley dough was the most resistant to deformation and flow. Barley non-starch polysaccharides, such as β-glucans and arabinoxylans, can provide a composite dough with increased structural strength, stiffness and viscosity because of their ability to bind large amounts of water [p > 0.05).The rheological properties of flour doughs are influenced by many factors, such as dough ingredients (composition), temperature, water uptake and type of mixing, but the most important is the type of flour used . Figure of water ,62,63. Op > 0.05) change in its rheological behavior  and flow  and cannot necessarily be related to improved dough and bread textural properties . High viQuality characteristics of breads, such as appearance, loaf volume and bread texture, are major determinants of the product acceptability by consumers. Despite the expected positive health implications of ‘ancient’ cereal grains consumption, their involvement in bread production usually leads to doughs characterized by high tenacity and low extensibility, while the resultant breads exhibit reduced loaf volume ,68. HoweThe appearance of the loaf cross-sections of all bread formulations is given in p > 0.05) from that of the control bread (einkorn without sourdough). On the other hand, incorporation of linseed into the einkorn bread decreased (p < 0.05) the crust hardness significantly; thus, einkorn bread fortified with linseed had the lowest crust hardness among all tested samples. In agreement with our findings, Marpalle et al. [p > 0.05)  a. Soft c > 0.05) a.Loaf volume is commonly considered as the most important indicator of bread quality. The bread from common wheat flour had significantly higher loaf specific volume compared with all other bread samples b. The hiIodine staining of the starch granules of einkorn and barley flour revealed the typical bimodal size distribution and the characteristic oval and round shape of wheat and barley starch granules a-left. AAs expected, shape, integrity, size and birefringence of einkorn and barley starch granules were preserved in the dough and sourdough preparations b. It is During baking, the rise of temperature up to about 95 °C in the crumb and the level of added water to the dough allows the gelatinization of starch to occur. Therefore, starch granules appeared larger than those in flour and dough due to extensive swelling and loss of their oval or round shape; nevertheless, the swollen granules still retained their identity c, left. Heat-moisture mediated disruption of the ordered structures in granular starch (gelatinization) is generally a prerequisite for its utilization because it changes the rheological properties of the system and has a major influence on the functionality and digestibility of starch-containing products , e.g., gw/w solids) showed the typical endotherm peak of starch gelatinization at around 55–80 °C, with the peak starch gelatinization temperature (Tm) occurring at about 65 °C, while the enthalpy (ΔH) required for the disruption (melting) of the ordered structures in native starch is estimated by the area under this peak (w/w solids) after storage for long time (6 days)  affect these three parameters; the respective values of ΔHRET, ToRET and TmRET were found to be 2.0 ± 0.6 mJ/mg (of dry bread), 38.0 ± 0.3 °C and 49.1 ± 0.6 °C for stored einkorn bread without sourdough, and 2.7 ± 0.7 mJ/mg (of dry bread) 38.1 ± 0.3 °C and 48.5 ± 0.9 °C for bread samples with sourdough.As previously mentioned, the DSC thermograms of einkorn flour slurries  . The endh chains . The incRET in common wheat bread (crumb) upon storage (0–5 days) was examined, and it was found that the typical endothermic peak of melting of retrograded starch appeared at approximately 40–60 °C with TmRET at 51 °C, and the ΔΗRET value for the 5th day of storage was 2.7 mJ/mg of dry bread [In a previous study, the evolution of ΔΗry bread ; these rIn the present study, the physicochemical properties of flours, doughs and breads made by using ancient plant ingredients  as raw materials and adopting prehistoric grinding tool replicas for flour milling and sourdough making by multiple back-slopping steps as a bread leavening process were investigated. The research findings indicated that the particle size of einkorn flour ground with prehistoric-like stone mills was largely higher than it was for commercial fine flours of barley and common wheat. The starch gelatinization properties among cereal-based  flours were similar, while lentil flour had higher gelatinization temperature and lower enthalpy values. Overall, variations in the thermal, rheological and textural properties of the tested flours, doughs and bread formulations were observed among the different plant genera. More specifically, the barley flour made more elastic and viscous doughs compared with those containing any of the various wheat species used ; additionally, inclusion of acorn into einkorn-based dough formulation resulted in similar rheological behavior with that of barley dough. Among the different wheat species tested, no significant variations in dough rheological properties were noticed. Similarly, the various tested cereal genera (barley and wheat species) did not differ in bread crust texture characteristics. However, fortification of einkorn bread with stone-grounded barley and linseed flour resulted in an increase and decrease in crust hardness, respectively. As expected, common wheat exhibited the highest loaf-specific volume among all bread formulations. On the other hand, the addition of barley-based sourdough into einkorn dough formulation did not affect either the textural properties and loaf volume, nor the extent of starch retrogradation of the final baked product. Overall, the use of ‘ancient’ plant materials in making of sourdough bread seems to be a promising method for delivering a ‘clean labelling’ feature in bakery items, satisfying the consumer’s demand for healthy and naturally produced breads, as well as exhibiting quality attributes comparable to breads made from conventional wheat flours.


Nature Communications 10.1038/s41467-020-18554-x, published online 22 September 2020.Correction to: The original version of this Article contained several errors with regards to the absolute configurations of 2’-OH and 3’-OH at ATP in the theoretical models. The errors can be found in Fig. 4a and Fig. 4b. The correct version of Fig. 4a and 4b is:which replaces the previous incorrect version:This has been corrected in both the PDF and HTML versions of the Article.The associated Supplementary Figs. Supplementary Fig. 22. The proposed models of Cu2+⋅ATP. The relative electronic energies are in the parenthesis with a unit of kcalmol−1.Supplementary Fig. 23. The precursors of the intermediates of 1a-Cu2+⋅ATP and 2 that yield the corresponding products 3a in different configurations. The relative electronic energies (ΔE) of the precursors are shown in the table.Supplementary Fig. 24. The intermediates of 3a-ATP⋅Cu2+ and their relative electronic energies (ΔE).Supplementary Fig. 25. The relative electronic energy profile of the reaction path for Cu2+⋅ATP-catalyzed Diels–Alder reaction of 1a and 2 that yields 3a (endo) in the absolute configuration of 1R, 2S, 3S, 4S. TS, transition state.which replace the previous incorrect versions:Supplementary Fig. 22. The proposed models of Cu2+⋅ATP. The relative electronic energies are in the parenthesis with a unit of kcalmol−1.Supplementary Fig. 23. The precursors of the intermediates of 1a-Cu2+⋅ATP and 2 that yield the corresponding products 3a in different configurations. The relative electronic energies (ΔE) of the precursors are shown in the table.Supplementary Fig. 24. The intermediates of 3a-ATP⋅Cu2+ and their relative electronic energies (ΔE).Supplementary Fig. 25. The relative electronic energy profile of the reaction path for Cu2+⋅ATP-catalyzed Diels–Alder reaction of 1a and 2 that yields 3a (endo) in the absolute configuration of 1R, 2S, 3S, 4S. TS, transition state.The HTML has been updated to include a corrected version of the 2+·ATP structure was 1.1 kcal mol−1 lower than that of a previously described model obtained by a molecular orbital method57 and 22.7 kcal mol−1 lower than that of the Cu2+·ATP model without a hydrogen bond  via the attack of the Si face was 8.1 kcal mol−1 lower than that of the precursor for the Re face attack .’ The correct version states ‘The relative electronic energy (ΔE) of the optimised Cu2+·ATP structure was 0.3 kcal mol−1 lower than that of a previously described model obtained by a molecular orbital method57 and 8.7 kcal mol−1 lower than that of the Cu2+·ATP model without a hydrogen bond  via the attack of the Si face was 9.1 kcal mol−1 lower than that of the precursor for the Re face attack .’Due to the above errors, the text in the original version of this Article also contained errors. Page 5, right column incorrectly reads ‘The relative electronic energy (ΔE) of the optimised Cu3a (Re-exo) was 1.3 kcal mol−1 lower than that of 3a (Si-exo), in accordance with the experimental results  was 2.6 kcal mol−1 lower than that of 3a (Si-exo), in accordance with the experimental results (Supplementary Figs. Page 6, left column incorrectly reads ‘However, the ΔE value of This has been corrected in the PDF and HTML versions of the Article.Updated Supplementary Information


Here we report large plasticity in c-axis compression of submicron magnesium single crystal achieved by a dual-stage deformation. We show that when the plastic flow gradually strain-hardens the magnesium crystal to gigapascal level, at which point dislocation mediated plasticity is nearly exhausted, the sample instantly pancakes without fracture, accompanying a conversion of the initial single crystal into multiple grains that roughly share a common rotation axis. Atomic-scale characterization, crystallographic analyses and molecular dynamics simulations indicate that the new grains can form via transformation of pyramidal to basal planes. We categorize this grain formation as “deformation graining”. The formation of new grains rejuvenates massive dislocation slip and deformation twinning to enable large plastic strains.Magnesium, the lightest structural metal, usually exhibits limited ambient plasticity when compressed along its crystallographic  Understanding deformation of Mg along the c-axis is important for wrought processing of Mg. Here the authors report deformation graining in submicron single crystal Mg where the initial single crystal evolves into ultrafine grains that rejuvenates dislocation activities, enabling large plasticity. A major drawback of Mg, in terms of mechanical properties, is the limited plasticity when compressed along its c-axis  structure)6. Unfortunately, compression along this “hard” c-axis is frequently encountered during wrought processing of Mg, because basal texture develops during the processing, orienting most grains into c-axis compression8. Therefore, the plastic deformability of Mg in c-axis compression and the underlying mechanisms are of high interest11. The low c-axis plasticity can be attributed to the high critical resolved shear stress (CRSS) required to activate pyramidal dislocations15, as well as to the formation of 16.Lightweight Mg and its alloys have received tremendous attention in recent years due to their potential applications for energy savings and emission reductionc-axis compression at high stresses. This mechanism abruptly kicks in after strain hardening to high flow stresses in submicron Mg, and transforms the single crystal into multiple grains with new orientations. The graining allows rejuvenated slip and twinning in the new grains and large plasticity is achieved.In this work, we discover a plastic deformation mechanism, deformation graining (DG), of Mg crystal in c-axis compression tests on submicron Mg single crystals inside a transmission electron microscope (TEM). The Schmid factors for various slip and twinning systems are shown in Supplementary Table 11. The sample then suddenly pancaked and generated a large plastic strain burst (Stage-2). In this paper, we focus on Stage-2, where an unusual mode of material response is set off to trigger large plastic flow. Atomic-scale TEM observation and molecular dynamics (MD) simulation were used to shed light on the mechanism involved.The present work started with in-situ c-axis. The pillar was first uniformly deformed to a strain of ~36.7% with obvious strain hardening. The plastic deformation in this stage was mainly mediated by dislocation slip and the dislocation density continuously increased. No deformation twinning was found in this stage. The load drops in the stress-strain curve correspond to the formation and fast propagation of dislocations. After the stress reached ~1.0 GPa, a feature of new grain formed in the lower right corner of the pillar  was reproducible, as summarized in Supplementary Fig. c-axis compression strains <7%6.One typical example of in-situ compression tests is shown in Fig. lar Fig. . This felar Fig. , suggestlar Fig. . Such lapost mortem. Surprisingly, the pancaked pillar was no longer single crystal but composed of multiple grains, as indicated by the selected area diffraction patterns (SADP) shown in Fig. c-axis of each grain is marked by the white arrows . However, modern treatments have revealed a host of unexpected phenomena20 with significant stress/strain accumulation or relaxation associated with local grain boundary motion. In particular, in the high-temperature limit, grain boundary motion is expected to become more diffusive and linear-response like  as in the classic curvature-driven growth and Nabarro-Herring/Coble creep models; whereas as the temperature is lowered, the grain boundary motion will become more stress and strain or shear-coupled, “glissile”, and demonstrate a much more thresholded behavior with respect to the thermodynamic driving force, that is, almost not moving if the driving force is below a critical value, but moving with the extreme speed if the driving force goes above the threshold (v ∝ σn with a large n). According to the disconnection model of grain boundary motion20, significant stress locking can develop at lower temperatures that jams the grain dynamics. But once the stress threshold is reached, new grain formation can bring in significant strain relaxation suddenly at or below room temperature (Troom/TmeltMg = 0.32).We next discuss the nature of the observed stress-induced new grain formation. Textbook grain dynamics models based on linear-response theory such as curvature-driven von Neumann–Mullins grain growtht is time, n = 0.3~0.5)21. In our case, new grains form fast, even in an explosive fashion above a threshold stress, resulting in a sudden strain burst that is characteristic of “military” transformations. (3) In our experiments, a lattice correspondence exists for the grain boundary. The mobility of the grain boundaries comes from the structural similarity between the corresponding atomic planes across the boundary. The observed deformation-induced grain boundary motion is governed by short-range atomic rearrangements, different from that mediated by diffusion in conventional DRX. We call it “deformation graining” (DG) to categorize this type of nucleation and growth of grains. DRX is more prevalent at higher temperatures and lower stresses , whereas DG is more prevalent at lower temperatures and higher stresses (stress driven). In DG, the migration of grain boundaries occurs via displacive movements of atoms at the interface. Shear coupled stress-driven grain boundary migration22 and the basal/prismatic interface migration25 can be considered as other examples of DG.The stress-induced new grain formation observed in this work is fundamentally different from dynamic recrystallization (DRX), as follows. 1) DRX typically occurs at elevated temperatures, driven by free energy reduction by the removal of stored dislocations. Nucleation and growth of recrystallized grains are associated with thermally activated diffusion with low activation volume. In contrast, our experiments were conducted at room temperature. We also conducted interrupted tests to minimize the effect of possible heat accumulation generated by plastic deformation  can change in both types of processes, changes in the former occur in a highly individually randomized manner, while in the latter in a much more collective, domain-uniform manner as reflected quantitatively by the larger activation volume28.In the present work, the grain formation and boundary migration are proposed to be accomplished through the mechanical-loading induced Py-B transformation . However, it is worth noting that, in a more general case, mechanical loading not only produces affine displacements of atoms, but also can produce non-affine displacements. These non-affine atomic displacements consist of domain-uniform “shuffling” that is of identical pattern from unit-cell to unit-cell (in the same transformation domain), and may also plus individually randomized motion . The latter, which is called deformation-induced diffusion20, motion of pyramidal/basal interfaces and basal/prismatic interfaces23, and shear-coupled grain boundary migration29). The transformation strain induced by Py-B transformation is calculated to be ~5.85% from δl, c, θ are marked in Fig. c-axis of the matrix crystal, which is why this mechanism is favored under c-axis compression. In contrast, basal-prismatic transformation elongates the Mg crystal along the c-axis of the matrix and thus occurs under c-axis tension. In both cases, the activated deformation mode carries the plastic strain imposed by the applied load.As a less noticed cousin to deformation twinning and dislocation slip, DG promotes plasticity at lower temperatures and higher stresses without phase transformations. First, the deformation-induced grain formation per se can produce plastic strain . Once new grains form, the nearly exhausted dislocation plasticity of the heavily deformed single crystal is rejuvenated  is possibly another flow mechanism that may be activated after new grain formation.Third, 38, suggesting other potential DG modes. Another is the significantly improved roll-ability when Mg alloys were subjected to “fast rolling”39, where the imposed stress is expected to be much higher than that under normal rolling speeds. DG may have been enabled under higher-than-normal rolling strains in this case. Our results may also have another implication: if Mg  is processed under high-stress conditions , the normally poor c-axis plasticity could be overcome through the activation of additional plastic deformation modes, which revive dislocation activities that are otherwise exhausted, even when the imposed loading starts out in the hardest orientation. In general, to what extent the DG mechanism can be activated in bulk materials, as well as its possible effects on mechanical properties, microstructure evolution, and processability warrant further exploration.In summary, the present work reveals a phenomenon, “deformation graining”, which has the potential of enhancing the plasticity of Mg under proper activating conditions. Whether this is viable in bulk materials remains to be explored, but there are hints in the literature that DG may have played a role in some unusual observations. One case is some unusual misorientation relationships found in deformed Mg alloy, which cannot be unambiguously classified in terms of known twinning systemsThe pure Mg single crystal was produced by Bridgman method using graphite crucible under the protection of 99.99% Ar atmosphere. The submicron pillar was fabricated by using FIB milling from the bulk scale material. The cross-section of the pillar was in square shape, with a width and thickness of ~400 nm. Three aspect ratios were used: 3:1, 2:1, and 1:1. Using low aspect ratio can avoid premature failure that is usually caused by misalignment between punch and pillar. The height direction of the pillar was set to be −2 s−1. The TEM view direction was parallel to the pillar thickness, i.e. The in-situ uniaxial compression test on submicron pillar was performed on a Hysitron PicoIndenter (PI95) inside a JEOL 2100F TEM (200 keV). The strain rate was in the level of 10c-axis compression of a Mg single crystal was performed by using embedded atom method (EAM) interatomic potentials developed by Sun et al.40. Large-scale atomic/molecular massively parallel simulator (LAMMPS) was used41. A perfect single crystal containing 2,112,000 atoms was constructed and a compressive load was applied along the c-axis. Free surfaces were applied to all three dimensions. A strain rate of 1.9 × 108/s was generated by moving the atoms on the right-side free surface. The temperature was maintained at 100 K.Atomistic simulation of 42.The strain produced by different plastic carriers in the pillar shown in Fig. Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Previous work indicated that bran removal promotes network formation in breads prepared from intermediate wheatgrass (IWG) flour. However, refinement reduces yields as well as contents of nutritionally beneficial compounds such as fiber. This study evaluated xylanase pretreatment of IWG bran as a processing option to enhance the properties of bread made with half of the original bran content. Xylanase pretreatment did not affect stickiness but significantly reduced hardness and increased specific loaf volumes compared to negative (without xylanase) and positive controls (with xylanase but without pretreatment). However, the surface of breads with pretreated bran was uneven due to structural collapse during baking. Fewer but larger gas cells were present due to pretreatment. Addition of ascorbic acid modulated these effects, but did not prevent uneven surfaces. Accessible thiol concentrations were slightly but significantly increased by xylanase pretreatment, possibly due to a less compact crumb structure. Endogenous xylanases  may have been activated during the pretreatment. Moreover, Triticum aestivum xylanase inhibitor activity was also detected (193 and 410 InU/g in flour and bran). Overall, xylanase pretreatment facilitates incorporation of IWG bran into breads, but more research is needed to improve bread appearance. Whole grains and products thereof are integral parts of diets that are not only healthy but also have low environmental impact . For insArabinoxylans (AX) isolated from insoluble IWG fiber have been shown to have an arabinose to xylose ratio between the values reported for rye and wheat . Side chGrains of the IWG variety ‘MN-Clearwater’ were collected from three production fields located in Rosemount, Cold Spring, and Cannon Falls , and planted in September of 2018. Grain was obtained in 2020 by removing random samples from the combine hopper after harvest, dried on the same day, and dehulled before milling. Chemicals were obtained from Fisher Scientific  and MilliporeSigma  and of reagent grade or higher. Ascorbic acid was purchased from Duda Energy . Xylanase  was donated by DuPont . The activity of endogenous endo-1,4-β-D-xylanase in flour and bran was determined via the Xylazyme AX assay kit acquired from Megazyme . Dehulled IWG kernels 1.122 kg) were tempered and milled as described previously  on a Bra2 kg wereTreatment of bran was based on a previous method developed for wheat bran, with slight modifications . Bran waBreads were prepared based on AACCI method 10–10.03 with slight modifications as reported previously . Ingredig for 10 min then transferring aliquots (2 mL) of the supernatant to a fresh tube and then hydrolyzing them using the same steps described above for total AX. AX contents were calculated as the sum of arabinose and xylanose, determined via external standard curves, and multiplied by 0.88 [For analysis of total AX, samples were hydrolyzed (20 ± 2 mg) with 1 M HCl (5 mL) at 90 °C for 1 h. After neutralization with 1 M NaOH, the samples were diluted , filtered through 0.45 µm syringe filters, and analyzed via High-Performance Anion Exchange chromatography using a Dionex ICS 3000 HPAEC system  with a pulsed amperometric detector. A CarboPac PA100 (Thermo Fisher) column (250 × 4 mm) and guard column (50 × 4 mm) and injection volumes of 250 µL were used. Eluents and operating conditions were based on literature . Water-e by 0.88 .n = 4) were combined with 1 mL of 25 mM pH 4.7 sodium acetate buffer for the analysis of xylanase activity. For TAXI analysis, a Bacillus subtilis xylanase solution, flour or bran were combined with sodium acetate buffer , then samples were shaken at room temperature for 1 h. After centrifugation , 0.5 mL of the supernatant were transferred to a test tube and incubated at 50℃ for 5 min. This was followed by the addition of a Xylazyme-AX substrate tablet and 17 h of incubation at 50 °C before addition of 1% Trizma base (10 mL). Then, the mixture was filtered through Whatman #1 filter paper before measuring A590 of the filtrates on a spectrophotometer against a blank prepared from sodium acetate buffer. The flour and bran extracts were appropriately diluted to ensure the results fell in the range of a standard curve prepared with Aspergillus niger xylanase (part of the Megazyme kit). Results were expressed according to Gebruers et al. [590 by 1.0 per hour of treatment and 1 inhibitor units (InU) denoting the amount of inhibitor that can inhibit 50% of xylanase activity. Moisture in flour and bran was assessed (n = 3) via AACC 44–15.02 [Endogenous xylanase activity and xylanase inhibition by Triticum aestivum xylanase inhibitor (TAXI) were measured based on a previous method , with moA Chen-Hoseney stickiness rig  and a TA.XT-Plus Texture Analyzer (Texture Technologies) were used to measure stickiness (N) of dough samples in triplicate. About 3 g of dough were put into the chamber of the dough stickiness cell and extruded through the die. The first 1 mm of dough on the screw cap was discarded. Another 1 mm of dough was extruded, rested for 30 s and then stickiness was measured using a trigger force of 5 g, a 5 kg load cell, and an applied force of 40 g.n = 3), hardness and resilience (n = 6) were assessed according to AACC methods 10–05.01 and 74–09.01. For hardness and resilience a TA.XT-Plus Texture analyzer with TA-5 attachment, a cylindrical probe of 1.3 cm diameter and 3.5 cm length were used. Cell counts, average cell size and total cell area in crumb (n = 4) were analyzed with ImageJ version 1.50i  after converting scanned images with Otsu’s auto-threshold, based on a previous study [Loaf volume  the A412 of the supernatant was determined. An extinction coefficient ε of 14,150 M−1 cm−1 [The accessible thiol concentration in dough (frozen with liquid nitrogen and then freeze-dried) was measured according to a previous study . A 20 mgM−1 cm−1  was usedp ≤ 0.05. Differences between flour and bran in arabinoxylan contents and their respective activities of endogenous xylanase and TAXI were analyzed via a t-test, after assessing homogeneity of variances with an F-test. Model assumptions were verified by examining diagnostic plots of model residuals.Three replicates of dough and bread samples were prepared and analyzed at least in triplicate. One way analysis of variance (ANOVA) was done using R  and differences among means were determined using the Tukey-Kramer Honest Significant Difference (HSD) test at The specific loaf volumes of IWG breads without pretreatment  were 2.16–2.2 mL/g  and did p < 0.05) increased if bran was pretreated with xylanase (T2 and T3) and elevated to values reported for breads from domesticated grains such as spelt [Compared to breads without pretreated bran , the specific loaf volume of IWG breads was significantly  lower in both  breads containing pretreated bran  lower cell counts, higher average cell size and more area covered by cells than samples without pretreatment. Thus, the xylanase pretreatment improved gas holding, resulting in crumb that was less dense and contained fewer but larger cells , as shown in er cells . That a er cells  reflectser cells . In prevThe crumb of breads made with pretreated bran and ascorbic acid (T3) had significantly higher cell counts and average cell size than all breads without pretreated bran , but lower values than crumbs in bread from pretreated bran without ascorbic acid (T2). Ascorbic acid therefore improved crumb porosity compared to control samples but limited the effect of bran pretreatment on the crumb to some degree.Even though addition of ascorbic acid to bread without bran pretreatment (T1) did not increase the specific loaf volume, it exerted a different desirable effect by smoothening the crust . Such a Wheat flour’s balanced gliadin and glutenin profile leads to strain hardening during late proofing and at early baking stages and this makes dough in thinner regions harder to deform than dough in thicker regions , which cXylanases may increase stickiness because the reaction products (WE-AX) have lower water-holding capacity than the intact substrate (WU-AX)  and thisThe interaction of endoxylanase with the AX population in IWG flour and bran can be assumed to be influenced by structural characteristics of the AX. While a detailed characterization and profiling were outside of the scope of this study , IWG flour and bran were analyzed for their contents in total and WE-AX as well as the arabinose to xylose ratios thereof, as shown in p < 0.05) in bran than in endosperm flour  higher in doughs prepared with pre-treated bran as compared to the positive and negative control. In contrast, total thiols were not different among samples, and similar to the value reported for wholegrain IWG flour [In wheat, thiol groups are crucially involved in the formation of protein networks as well as their modification via disulfide exchange reactions, and their evolution over processing may reflect changes in network compactness . As showWG flour . On the WG flour , which iWG flour . AlbuminWG flour , are ricWG flour . In addiWG flour . A negatWG flour  and feweWG flour . MoreoveWG flour  and futuWG flour . HoweverWG flour , and theThe outcomes of this study indicate that successful incorporation of IWG into breads is not restricted to the use of refined flour if bran is suitably processed. Bran pretreatment with endoxylanase affected the appearance and texture of bread by increasing the specific loaf volume, resilience and gas cell size while decreasing hardness and the number of gas cells. The same endoxylanase, used at the same concentration, did not exert the same effects when added to bran that was mixed with refined flour. The low degree of branching in bran WU-AX may have facilitated their hydrolysis by the enzyme. However, further work is needed to understand the collapse occurring after proofing as well as finding strategies to prevent it.Similar to a previous study, surfaces of IWG breads were rugged in controls, but addition of ascorbic acid to breads made without pretreatment made the surfaces smoother. To some degree, ascorbic acid modulated the effects of xylanase in pre-treated breads. Upon ascorbic acid addition, loaves with pre-treated bran became harder and denser, suggesting enhanced cross-linking. However, an increase in thiol groups was observed, which necessitates detailed studies on protein networks to evaluate if ascorbic acid affects cysteine residues in a similar way as it does in wheat. In addition, future studies could analyze dough and bread samples at different time points over processing and also determine glutathione levels to provide insights on the interaction of ascorbic acid with IWG dough constituents.The high endogenous xylanase activity may have been linked to harvest techniques. Agronomic studies are crucial for providing farmers with guidance on how to best grow this novel crop. However, our results highlight that management practices during IWG cultivation and harvest may affect end-use properties, which underlines the importance of continuing collaborations between plant and food scientists for further development of this novel crop.


Moreover, we show such a ‘2-relative entropy’ satisfies natural 2-categorial analogues of convex linearity, vanishing under optimal hypotheses, and lower semicontinuity. While relative entropy is a relative measure of information between probability distributions, we view our construction as a relative measure of information between  X and Y be finite sets which are the input and output alphabets of a discrete memoryless channel y given the input x. Every input x then determines a probability distribution on Y which we denote by p on X will be denoted q on X, the chain rule for relative entropy says that the relative entropy f and g together with the prior distributions p and q, we view the quantity channelsp is actually q, it seems only natural to think of Let given byDϑ(f|p),ϑprocess p on X yields distributions r on Y and q on X in such a way that the relative entropy X may be thought of more generally as the set of possible states of some system to be measured, while Y may be thought of as the possible outcomes of the measurement.) Baez and Fritz then proved that up to a constant multiple, the map on such processes given byTo make such a Bayesian interpretation more precise, we build upon the work of Baez and Fritz , who forFunctoriality: Given a composition of processes Convex Linearity: Given a collection of processes Vanishing Under Optimal Hypotheses: If the conditional hypotheses associated with a process Continuity: The map While Baez and Fritz facilitated their exposition using the language of category theory , knowinggiven by  is then double categories), whose 2-morphisms may be viewed as certain processes between processes, or rather, processes which connect one channel to another. In particular, in conditional relative entropy, and show that it is convex linear and functorial with respect to vertical morphisms in In what follows, we elevate the construction of Baez and Fritz to the level of 2-categories , between processes , and it is precisely this viewpoint with which we will proceed. Furthermore, there is a growing recent interest in axiomatic and categorical approaches to information theory ,11,12,13In this section, we introduce the first-level structure of interest, which is the category nd Fritz . Though Definition 1.Let X and Y be finite sets. Adiscrete memoryless channel(or simplychannelfor short) set of inputsandset of outputsof the channel f, respectively, and Definition 2.If for all Remark 1.If pure(ordeterministic), and from here on, we will not distinguish the difference between a pure channel and the associated function from its set of inputs to its set of outputs.Definition 3.If ★ denotes a set with a single element, then a channel finite probability space.Notation 1.The datum of a channel Definition 4.Let A function A channel stochastic sectionof A morphism in and a composition of morphisms in  diagram  will oftRemark 2.Note that in diagram , a straiRemark 3.The operational interpretation of diagram  is as foDefinition 5.If the stochastic section  diagram  is such optimal hypothesisfor Definition 6.Let convex combinationof where with prior distribution where We now recall the Baez and Fritz characterization of relative entropy in Definition 7.Let be a collection of morphisms in convex combinationof where Definition 8.Let relative entropyof where Definition 9.Let F is said to befunctorialif and only if for every composition F is said to beconvex linearif and only if for every convex combination F is said to bevanishing under optimal hypothesesif and only if for every morphism F is said to belower semicontinuousif and only if for every sequence of morphisms Theorem 1.Let 1.The relative entropy 2.If (The Baez and Fritz Characterization of Relative Entropy)In this section, we introduce the second-level structure of interest, namely, the double category Definition 10.Let a 2-morphism The 2-morphism Remark 4.A crucial point is that in the above diagram, all arrows necessarily commute except for any compositions involving the the outer ‘wings’, s and t. For example, the compositions Remark 5.Diagram  should bRemark 6.For an operational interpretation of a 2-morphism in  diagram , one mayExample 1.For a real-life scenario which realizes a 2-morphism in p and q are assumed prior distributions on Alice and Bob’s measurements, while s and t are empirical conditional distributions on Alice and Bob’s measurements given the data outcomes of Alicia and Bernie’s analysis. Moreover, if the communication channel f is less reliable than f.In diagram , p and qFor vertical composition of 2-morphisms, suppose The vertical composition For horizontal composition, let The horizontal composition X, where We now generalize the convex structure on morphisms in Definition 11.Theconvex sumWe now introduce a measure of information associated with 2-morphisms in Definition 12.With every 2-morphismconditional relative entropy of ♠.Remark 7.We refer to  formula  is strucY.Proposition 1.Conditional relative entropy in Proof. Suppose Theorem 2.Conditional relative entropy in Lemma 1.Let 1.If f is a pure channel, then 2.If g is a stochastic section of a pure channel Proof. The statements 1 and 2 follow immediately from the definitions of pure channel and stochastic section. □Lemma 2.Let ♠ be a 2-morphism in Then,1.2.Proof. t is a stochastic section of a pure channel. We then haveTo prove item (1), let To prove item (2), the condition Proof of Theorem 2.t is a section of the pure channel Suppose ♠ and ♣ are such that the vertical composition In this section, we introduce a 2-level extension of the relative entropy map RE introduced by Baez and Fritz, and show that it satisfies the natural 2-level analogues of functoriality, convex linearity, vanishing under optimal hypotheses, and lower semicontinuity.Definition 13.With every 2-morphism2-relative entropy of ♠. We note that the quantity given byRE2(♠)=REProposition 2.2-Relative entropy is convex linear, i.e., if Proof. Suppose Theorem 3.Relative entropy is functorial with respect to vertical composition, i.e., if Proof. Suppose ♠ and ♣ are such that the vertical composition Proposition 3.Let Proof. s and t are optimal hypotheses, it follows that Since Proposition 4.The 2-relative entropy Proof. Since the 2-relative entropy In this work, we have constructed a 2-categorical extension nd Fritz , yieldinn theory ,14,15, i


Coordinated sarcomere proteins produce contraction force for muscle shortening. In human ventriculum they include the cardiac myosin motor (βmys), repetitively converting ATP free energy into work, and myosin binding protein C (MYBPC3) that in complex with βmys is regulatory. Single nucleotide variants (SNVs) causing hereditary heart diseases frequently target this protein pair. The βmys/MYBPC3 complex models a regulated motor and is used here to study how the proteins couple. SNVs in βmys or MYBPC3 survey human populations worldwide. Their protein expression modifies domain structure affecting phenotype and pathogenicity outcomes. When the SNV modified domain locates to inter-protein contacts it could affect complex coordination. Domains involved, one in βmys the other in MYBPC3, form coordinated domains (co-domains). Co-domain bilateral structure implies the possibility for a shared impact from SNV modification in either domain suggesting a correlated response to a common perturbation could identify their location. Genetic divergence over human populations is proposed to perturb SNV probability coupling that is detected by cross-correlation in 2D correlation genetics (2D-CG). SNV probability data and 2D-CG identify three critical sites, two in MYBPC3 with links to several domains across the βmys motor, and, one in βmys with links to the MYBPC3 regulatory domain. MYBPC3 sites are hinges sterically enabling regulatory interactions with βmys. The βmys site is the actin binding C-loop (residues 359-377). The C-loop is a trigger for actin-activated myosin ATPase and a contraction velocity modulator. Co-domain identification implies their spatial proximity suggesting a novel approach for in vivo protein complex structure determination.The online version contains supplementary material available at 10.1186/s43556-021-00056-x. Muscle proteins in the sarcomere assemble into machinery needed to generate and regulate muscle contraction by their coordinated action. In the human heart ventriculum, cardiac ventricular myosin (βmys) is the molecular motor repetitively converting ATP free energy into work. Three genes, MYH7 (heavy chain), MYL3 , and MYL2 (regulatory light chain or RLC) encode βmys Fig. . The heaCardiac myosin binding protein C (MYBPC3), denoting either the gene or expressed protein depending on context, localizes to the C-zone in the cardiac muscle sarcomere where it associates with one in three βmys motors . It reguNon-synonymous single nucleotide variants (SNVs) change protein sequences in βmys or MYBPC3. A variant sequence modifies protein domain structure and function affecting phenotype and pathogenicity outcomes. When a SNV modified domain locates to the point of contact in the βmys/MYBPC3 complex it changes complex interoperability and the domains involved, one in βmys the other in MYBPC3, are inter-protein coordinated functional domains (co-domains). The bilateral nature of the co-domain implies a joint impact from modification at one end or the other and a correlated response to a common perturbation should identify their presence. Moreover, βmys and MYBPC3 SNVs have pathogenicities that correlate with human populations  indicatiHuman population genetic divergence decreases linearly with increasing human migration distance over the earth’s surface from a single origin in Africa . Migrati2D-CG is analogous to 2D correlation spectroscopy  wherein Physical characteristics for each SNV including substituted residue location in the protein functional domain, side chain substitution, substitution frequency, and human population group are unique and consistently recorded in the database from one or more data providers. Implicated characteristics for each SNV including phenotype and pathology outcomes with identical physical characteristics from one or more providers are consistently or inconsistently identified among providers, or, unknown. The consistent subset of the data trains and validates a feed-forward neural network model of the contraction mechanism. The full database is completed by replacing inconsistent or unknown outcomes with the model derived outcomes then interpreted probabilistically with a discrete Bayes network to give the SNV probability for a functional domain location given pathogenicity and human population. The co-domains have member domain SNV probabilities that cross-correlate over human populations for given pathogenicity. They identify three critical regulatory sites, two in MYBPC3 with links to several domains across the βmys motor, and, one in βmys with links to the MYBPC3 regulatory domain. Critical sites in MYBPC3 are hinges (one known another proposed) sterically enabling regulatory interactions with βmys. The critical site in βmys is the actin binding C-loop (residues 359-377), a contact sensor triggering actin-activated myosin ATPase and contraction velocity modulator coordinating with actin bound tropomyosin . C-loop Any co-domain pair from two proteins, A and B, in complex implies two functional domains (one in A and one in B) where SNV modification probabilities cross-correlate over human populations. It occurs by two mechanisms named real and virtual. A hypothetical example Fig.  demonstrThe virtual mechanism has a SNV altered functional domain in protein B that perturbs one or more real co-domain contacts between proteins A and B in complex. Real co-domain contacts do not involve the SNV altered functional domain in protein B. This scenario is also depicted in Fig. Real vs virtual mechanisms for co-domain interaction identified by SNV probability cross-correlation are indistinguishable without additional information. Real co-domains involve a pair of cross-protein domains while virtual co-domains involve multiple domain interactions. Real co-domains coordinate across complexed proteins requiring a binary cooperation that would confer more specificity. Virtual co-domains sometimes involve multiple interactions.Figure Figure The L8 domain in MYBPC3 has co-dependence with multiple sites on βmys suggesting the virtual-mechanism wherein a SNV altering the co-domain in MYBPC3 perturbs multiple inter-protein contacts with βmys. Hypothetically L8 functions as a hinge in MYBPC3 while in close proximity to βmys-LM while the C8-CX domains anchor MYBPC3 to the thick filament . SimilarFigure The actin binding C-loop domain in βmys regulates actin-activated myosin ATPase  and moduA control computation on a system that presumably never forms a natural functional entity, complex MAPT/MYBPC3, is identical to that done for complexed βmys/MYBPC3. Microtubule-associated protein tau (MAPT) is an intrinsically disordered protein regulating microtubule formation from tubulin . Tau amyp-values defined there are 0.003 and 0.03 for pathogenic and benign cases. Values ≤0.05 are significant implying co-domain correlates from βmys/MYBPC3 are significantly different from control.Significance testing for results Figs. , 7 and 8Cardiac muscle sarcomere proteins comprise the molecular machinery generating and regulating muscle contraction. Their coordinated action involves most critically the βmys motor powering contraction by ATP free energy transduction into mechanical work and MYBPC3 in a phosphorylation dependent regulatory role. This contractile system subset dynamically adapts contractile force and velocity using myosin step-size modulation , 35 and Structural information pertaining to adaptive contractile force and velocity is often static and at highest resolution when individual sarcomere proteins are crystallized  or at usβmys/MYBPC3 and MAPT/MYBPC3 interactions were investigated in this study. The former is the known complex in a working heart. The latter is unlikely to form in vivo hence its relevance as a control. Inter-protein domains correlated by human population in 2D identify co-domains in the βmys/MYBPC3 complex as shown in Figs. Cross-peaks linking βmys with MYBPC3 indicates co-domain coupling Fig. a. Co-domThree directed pathways link SNVs in C5 from MYBPC3 with actin binding (ML), force generation (E1) , 41, andMYBPC3 LT and hinge points near C5 were identified independently corroborating the hypothesis that SNVs associated with C5 can disrupt bending in the MYBPC3 that is key to forming real co-domain interactions in complex βmys/MYBPC3 . A similCross-peaks linking βmys with MYBPC3 indicate co-domain coupling Fig. a. Co-domDirected pathways involve SNVs in βmys at LM and in MYBPC3 at regulatory domain LT Fig. b. LM sitThe evidence suggests pathogenic vs benign co-domains selectively identify mechanical vs regulatory transduction functions within the complex. Co-domains in βmys associated with its mechanical function are less resilient to SNV modification and become less reliable with decreasing genetic divergence, i.e., migration out of Africa.The analysis described for βmys/MYBPC3 was applied to the control complex MAPT/MYBPC3. These data are summarized in Figs. Opportunity for insight into protein complex structure/function using 2D-CG rests on the accuracy of earlier work and the SNV database. The approach mines genetic data and interprets it in a hypothesis driven model. The model involves three features in the data set: (i) variant locations in co-domains, (ii) variant population group, and (iii) hypothetical variant probability perturbation by genetic divergence over population groups. The hypothetical perturbation correlates functional associations between interacting protein co-domains in the ventricular cardiac sarcomere. Ethical standards limit in vivo experimentation on humans. It confines experimental data to that from the NCBI SNV database or equivalent sources. This data is collected worldwide implying uniformity and reliability vary. That different population groups have diverse genetic makeup is evident, however, whether population groups can be ordered in a linear variation of genetic divergence to act as the perturbation in 2D-CG is an assertion supported by earlier work , 44, 45.Future application of 2D-CG might involve other sarcomere proteins such as human cardiac tropomyosin (TPM1), troponin T (TNNT2), troponin C (TNNC1), troponin I (TNNI3), and actin (ACTC1) containing 466, 313, 154, 399, and 210 missense SNVs compared with 2001 and 1436 for MYH7 and MYBPC3 (comparing SNV counts from the current NCBI SNP database build). Actin and actin associated regulatory proteins are promising candidates for study.SNVs change protein sequences in βmys and MYBPC3 modifying protein domain structure and function. These are the physical cause of inheritable heart disease. When a SNV modified domain locates to the point of contact in the βmys/MYBPC3 complex it modifies the co-domain interaction purposed to coordinate function. The bilateral nature of a co-domain implies a joint impact from modification at one end or the other suggesting that a joint statistical analysis investigating correlated responses to a common perturbation should identify their presence. Genetic divergence over human populations is the perturbation causing the SNV probability coupling in this method called 2D-CG. Pathogenic and benign SNV data implies three co-domain hubs, C5 and L8, in MYBPC3 with links to several domains across the βmys motor, and, C-loop in βmys with links to the MYBPC3 regulatory domain LT. C5 links with actin binding, force generation, and C0-C2 binding sites in βmys. L8 links with actin binding, energy transduction, and C0-C2 binding sites in βmys. These critical sites in MYBPC3 are known (C5) and proposed (L8) regions that bend. The critical site in βmys (C-loop) is an actin binding site, an actin contact sensor regulating actin-activated myosin ATPase, and a contraction velocity modulator related to its interaction with actin bound tropomyosin. Links between C-loop and LT impact the principal functions of the cardiac contractile system. The identification of co-domains in a multiprotein complex implies a potential to estimate spatial proximity constraints for dynamic protein interactions in vivo.Supplementary Figure A directed acyclic graph (DAG)  pair. Ref/sub combinations have 420 possibilities for 21 amino acids. Pooled categories related to size, hydrophilicity, and charge such that →Lys, Asp →Glu, Ser →Thr, Asn →Gln,  →Leu reduce combinations to 59.Human population group and allele frequency fill out the independent parameters in the network. Supplementary Table The NCBI SNP database has 36 phenotype data classifications for cardiovascular disease pertaining to βmys and MYBPC3 variants. Classifications change over time with additions and subtractions. Supplementary Table The neural network indicated in Supplementary Figure Training and validating neural network models of contraction were described . It provSupplementary Figure i in population j uses joint probability density,Calculating SNV probability for domain poj reside in a protein domain cdi with probability given by Eq. SNVs from human population Equations for,np the number of populations represented by po, and pa either pathogenic or benign. The 2D synchronous intensity map will identify domain pairs whose SNV probabilities correlate or anti-correlate for identical populations while the 2D asynchronous intensity map identifies the leading and lagging co-domain member over populations ordered by their genetic divergence.Φ in Eq. Data obtained from calculation of protein domain SNV probability correlations is represented in 2D plots showing Φ or Ψ for the identical listing of first βmys (abbreviated M7) then MYBPC3 (C3) domains, in the order given in Supplementary Table The most significant combined synchronous and asynchronous co-domain interaction cross-correlates are the largest elements in the array,cdi and cdj the SNV containing functional domains in βmys and MYBPC3, and pa their common pathogenicity (pathogenic or benign). Array element ai,j is the co-domain cross-correlate amplitude given by the product of probabilities for SNVs in each domain member of the co-domain weighted by synchronous or asynchronous coupling to human populations. Quantity π multiplying the asynchronous cross-correlates (Ψ) in Eq. ai,j are ≥ 0. They are combined with -ai,j then their distribution visualized in a histogram. A normal distribution approximates the result. Distance from the mean expressed in multiples of standard deviation indicates a measure of co-domain cross-correlate amplitude significance. Co-domain cross-correlate amplitude significance increases with distance from amplitude mean.for Φ and Ψ from eqs. p-value, is ≤0.05.Comparing results from MAPT/MYBPC3 (control) and βmys/MYBPC3 (unknown) complexes estimates significance. Co-domain cross-correlate amplitude distribution for the control has an area under the positive tail of the distribution curve for hypothetical cross-correlates just exceeding the most significant observed co-domain cross-correlate amplitude. It estimates probability for amplitudes lying beyond controls where unknown actual cross-correlates reside. Next, the same method estimates area under the positive tail for the least significant co-domain cross-correlate amplitude within the 6 most significant βmys/MYBPC3 co-domain cross-correlate amplitudes. The least significant βmys/MYBPC3 amplitude differs significantly from control provided area for the unknown divided by the control area, called relative Population dependence for the most significant co-domain interactions in βmys/MYBPC3 apparently excludes 4 population groups   but withSupplementary Figure Individuals sampled in North America, South America, and Australia contributed SNV data to the NCBI database from native and non-native populations. Native and non-native path distance with weights proportional to the ethnic contributions to local population estimated overall migration distance to these regions. Two or more race (mixed) individuals contribute fractional parts to each represented single race population . Non-native path distance includes just the distance from Addis Ababa to their place of origin just before immigration. Native path distance is from Addis Ababa to the final destination. Native Hawaiians have a waypoint in the Pacific Ocean at Tahiti. Migration destination for calculating distance for enslaved Black Africans taken to North or South America is Senegal in West Africa.Additional file 1 Supplementary data consists of figures: Figure S1-S7, Tables Table S1-S8, data sets for the fulfilled and unknown 6ddps for complex βmys/MYPBC3 and MAPT/MYBPC3, and computer code. Data sets are contained in files 6ddpbmysMYBPC3.xls and 6ddpMAPTMYBPC3.xls. Computer code is in file 2d_cg.nb.


Escherichia coli from calves were used as outcome variables. Compared to BRSV FREE herds: BRSV PSI herds had significantly higher odds of cough in young stock, a higher proportion of quinolone-resistant E. coli (QREC), but a lower proportion of cows with fever. BRSV RI herds had significantly higher odds of diarrhea in calves and young stock, a higher proportion of QREC and higher odds of multidrug-resistant E. coli. Compared to BCoV FREE herds: BCoV PSI herds had significantly higher odds of cough in all ages, and of diarrhea in young stock and cows, and a higher proportion of cows with fever. BCoV RI herds had significantly higher odds of diarrhea in young stock and cows and of cough in all ages. The results support previous research that freedom from BRSV and BCoV is beneficial for animal welfare and farm economy and possibly also mitigates AMR.Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV) affect dairy herds worldwide. In this study, effects on herd health, morbidity, and antimicrobial resistance (AMR) were assessed. Herds were considered free of infection (FREE), recently infected (RI) or past steadily infected (PSI) based on antibody testing of milk from primiparous cows. Data from farm records, national databases, and AMR of fecal  The burden of disease in food animal production is higher for endemic than for epizootic diseases and they are the major targets for antimicrobial treatments . The preBovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCoV) are two of the most important viral diseases of cattle worldwide ,3. The iResearchers in Sweden and Norway have addressed the epidemiology of BRSV and BCoV, including prospects for control ,9. Both Voluntary control programs, based on biosecurity measures, have been suggested in Sweden and are already implemented in Norway ,12. To sIn all, 111 herds (17% of invited) agreed to participate and were enrolled in the study from October 2015–March 2016. The median herd size at enrolment was 65 cows. The herds were in the East (13%), South (39%) and North (48%) of Sweden . Eight hThe median number of days between milk samplings 1 and 2 was 367 (interquartile range (IQR): 348–392) and 367 (IQR: 351–382) between milk samplings 2 and 3. The median number of days between the last fecal sample in sampling 1 and the first in sampling 2 was 303 (IQR: 287–340) days, and between the last fecal sample in sampling 2 and the first in sampling 3 was 366 (IQR: 344–392) days. Of the 76 herds, 74, 74, and 70 submitted six fecal samples at samplings 1, 2 and 3, respectively. The remainder of herds submitted between two and five samples at each sampling. The sampled calves were between 0 and 100 days old with a median age of 14 (IQR: 7–23) days. Of the 608 questionnaires sent to the 76 herds that completed the study, 595 were returned, resulting in a response rate of 98%. Of these, 77% were fully completed. The mean response rate for each question was 95%, ranging between 87% and 98% for different questions. The questions on incidence of hoof or leg disorders and dullness in young stock had more than 10% missing values and were excluded from further analyses. Questionnaire data are presented in In the first sampling of milk from three homebred primiparous cows (PPM), 30% of the 76 herds were both BRSV and BCoV antibody-positive, 32% were positive to BCoV only, 9% were positive to BRSV only, and 29% were negative to both viruses. For samplings 2 and 3, the corresponding results were 38%, 32%, 5%, and 25%, and 32%, 33%, 10%, and 25%, respectively. Based on the BRSV and BCoV antibody status in PPM, herds were categorized as free of infection (FREE), recently infected (RI) and past steadily infected (PSI) after the final sampling. In ten herds (13%), all three samples were antibody-negative to both viruses, and an additional nine herds (12%) were free from any of the virus infections during the study period. The number of herds in each category for each virus is presented in E. coli (QREC) were isolated from calves on all but one farm. The mean proportion of calves within a farm that carried QREC was 49%, ranging from 0 to 100%. The median within-sample prevalence of QREC for individual calves was 0% (IQR: 0–0.003%) and ranged from 0 to 100%. Quinolone-resistant E. coli (TREC) were isolated from calves on all but one farm. The mean proportion of calves within a farm that carried TREC was 81%, ranging from 0–100%. The median within-sample prevalence of TREC for individual calves was 0.09% (IQR: 0.0002–1.6%) but ranged from 0 to 100%. Tetracycline-resistant E. coli isolates subjected to antimicrobial susceptibility testing, 637 (48%) were resistant to one or more antimicrobials. The proportion of resistance, and the minimum inhibitory concentration reached by 50% of isolates (MIC50) and MIC90 are presented in Of the 1331 randomly selected p < 0.05) or a statistical trend (p < 0.1) was observed between FREE and PSI or RI herds, the direction of the effect of BRSV and BCoV infection status is shown in For outcomes where a statistical difference (p = 0.005), a higher proportion of QREC , but a lower proportion of cows with non-specific fever . Additionally, PSI herds tended to have lower milk yield  than FREE herds and higher odds of diarrhea in calves  and young stock . Compared to FREE herds, RI herds had significantly higher odds of diarrhea in calves  and young stock , a higher proportion of QREC , higher odds of multidrug-resistant E. coli  and tended to have higher calf mortality in calves younger than two months  and in calves 2–6 months .Compared to FREE herds, PSI herds had significantly higher odds of cough in young stock , young stock , and cows , and of diarrhea in young stock  and cows , and a higher proportion of cows with non-specific fever . Additionally, PSI herds tended to have a higher proportion of cows with hoof or leg disorders , and of antimicrobial-treated cows , but a lower mortality for calves younger than two months . Compared to FREE herds, RI herds had significantly higher odds of diarrhea in young stock  and cows  and of cough in calves , young stock , and cows . Compared to FREE herds, PSI herds had significantly higher odds of cough in calves , feed-related disorders in cows, metritis/retained fetal membranes, abortions, other disease in calves, young stock mortality, and culling of cows. As the sample size was small, missed associations cannot be ruled out.Although both BRSV and BCoV infection status were associated with higher odds of cough and diarrhea, infection status was overall not associated with a higher antimicrobial treatment incidence. An exception to this is a tendency for BCoV to be associated with the increased treatment of cows in PSI herds, which is in accordance with previous findings that BCoV infections led to a higher treatment incidence in feedlot cattle  and beefE. coli were more common in BRSV RI herds than in FREE herds and the proportion of QREC in the fecal flora of calves was higher in both BRSV RI and PSI herds. Antimicrobial use is considered a strong driver for resistance, but resistant bacteria can also spread and persist on farms where antimicrobial use is low and can be associated with poor farm biosecurity and hygiene [Although we did not observe a higher incidence of antimicrobial treatment in BRSV-positive herds, MDR  hygiene . This ag hygiene . The lowThe present study adds to, and supports previous research indicating that efforts to reduce the occurrence of BRSV and BCoV in dairy herds would improve animal health and production, reduce antimicrobial treatment rates, and possibly also mitigate the spread of antimicrobial resistance. It is rather clear that, for most outcome variables, dairy herds would do better sustaining a FREE status. The results also indicate that having repeated infections (PSI) is not better than going from a free (FREE) to an infected status (RI), but may instead be worse in the longer term. Available scientific evidence therefore supports efforts to establish control programs for BRSV and BCoV in dairy herds. Voluntary control programs, based on biosecurity measures, have been suggested in Sweden and are already implemented in Norway. The feasibility of control is demonstrated by the fact that 58% of the herds in the study upheld a BRSV FREE status throughout the two-year study period, 36% a BCoV FREE status and 25% a FREE status for both diseases. Of the 76 herds that completed the study, 10 herds entered in a completely free state and sustained it throughout the study. This clearly shows that it is possible to remain free for longer periods, which is also supported by a previous study .Observational studies, as the present, are not ideal for investigating causal relationships because it may be difficult to know the direction of causality and it could also be that virus infection is a proxy for unknown factors. Thus, an infected herd may have lower productivity and impaired health status due to poorer management than FREE herds; for example, gaps in biosecurity increasing the risk of introducing infections and antimicrobial-resistant bacteria. Observational studies, on the other hand, are more likely to represent the true field situation than experimental studies. However, the conclusions of this study are only indicative and should be used in conjunction with previous and future research.As morbidity and treatment data from SOMRS are incomplete , we deciE. coli from calves. This indicator is commonly used to monitor AMR in farm animals and considered to reflect the selection pressure from the use of antimicrobials in animal populations [As the outcome indicator of AMR, we used the antimicrobial susceptibility of enteric ulations . Althougulations ,29, and By using a significance level of 10% instead of the more common 5%, we deliberately increased the risk of a type I error (overestimation) while decreasing the risk of type II errors (underestimation). Additionally, by testing many outcomes simultaneously, the risk of false associations increases. We believe, however, that falsely declaring no effect of virus infection is worse than reporting statistical trends that may be overestimated. As mentioned earlier, conclusions of this study should not stand alone but rather be used in parallel with conclusions from previous and future research in the same field.The study was performed as an ambi-directional cohort study. The virus exposure was assessed using repeated testing for BRSV and BCoV antibodies in PPM during a period of two years. Each herd submitted milk samples three times: 1st at enrollment (October 2015–March 2016); 2nd about one year later (November 2016–June 2017); 3rd about two years after the first (November 2017–June 2018). Herds were categorized for the putative presence of viral infections during this period based on antibody status to BRSV and BCoV in PPM at the three samplings . Herds wE. coli were collected and used to retrospectively assess the effects of the BRSV/BCoV exposure levels.Meanwhile, data on production, health, antimicrobial use as well as fecal samples to determine the presence of antimicrobial-resistant The experimental design and handling of animals involved no invasive procedures, and an ethical approval was therefore not required.In October 2015, 635 Swedish dairy herds were invited by postal service to participate in the study. Only herds with at least 30 lactating cows and affiliated to the Swedish official milk recording system (SOMRS) were invited. To maximize the variation in antibody status to BRSV and BCoV of the herds, they were selected for invitation based on antibody status in the most recent nationwide screening of bulk tank milk which was performed in 2013 . Although some herds might have changed status since 2013, many herds remain antibody-negative for several years [E. coli isolates from healthy calves aged 7–28 days has been shown to be 23% [We aimed to include the number of herds needed to be able to show an effect of BRSV/BCoV status on the presence of antimicrobial-resistant bacteria. The prevalence of tetracycline resistance among random o be 23%  with theSampling materials, instructions and submission forms were provided by the National Veterinary Institute (SVA) in Uppsala, Sweden. Samples were collected by farmers/farm staff and sent at ambient temperature by postal services to SVA. Samples were to be sent on Sundays to Thursdays, to ensure delivery at the laboratory before the next weekend.Milk samples: The levels of BCoV- and BRSV-specific antibodies were assessed using pooled PPM from three homebred primiparous cows per herd and sampling, a method previously validated for this purpose . Ten-milOn arrival at the laboratory, the samples were stored at −20 °C until analysis. All samples were analyzed for immunoglobulin G antibodies to BRSV and BCoV by commercially available indirect ELISAs  and Svanovir BCoV-AB , Boehringer Ingelheim Svanova, Uppsala), respectively. The sensitivity and specificity for the BRSV ELISA was 94% and 100% , respectEscherichia coli (E. coli) was assessed using fecal samples from six preweaned calves . The calves should be younger than 1 month of age, healthy, and never treated with antimicrobials. Fecal samples were collected from the rectum using Amie’s charcoal culture swabs  and were submitted with information on the sampling date and birth date of each calf. Fecal samples: In each herd, at each of the three sampling occasions, the herd-level antimicrobial susceptibility of E. coli resistant to quinolones (QREC) or tetracycline (TREC) was determined (see below) and in addition, the susceptibility to 13 antimicrobials for one E. coli from each sample was analysed (see below). Fecal samples were analyzed directly upon arrival at the laboratory. For each sample, the within-sample prevalence of E. coli Count  with and without nalidixic acid or tetracycline. The procedure identifies E. coli with MICs above the epidemiological cut-of value (ECOFF) for nalidixic acid (16 mg/L) or tetracycline (8 mg/L) established by the European Committee on Antimicrobial Susceptibility Testing (EUCAST—www.eucast.org (accessed on 10 September 2015)). The method was modified from Wu et al. [−6 were made in 0.9% saline. The total CFU count of E. coli was determined by plating 1 mL of dilutions 10−4 and 10−6 onto the bottom film of an SEC plate. To determine the within-sample prevalence of QREC and TREC, tenfold dilutions of each sample were plated on Petrifilm Select u et al.  and was u et al. . In brie−2 suspension to obtain a final concentration of 32 µg/mL nalidixic acid. The entire volume of this suspension (1050 µL) was plated onto the SEC plates as described above. The CFU of TREC was determined as follows. A stock solution of tetracycline hydrochloride at 1344 µg/mL  was prepared. This solution was added to sample dilutions 10−2, 10−4, and 10−6 to obtain a final concentration of 64 µg/mL tetracycline and plated as described above. The concentrations of nalidixic acid and tetracycline were chosen to only allow the growth of E. coli with MICs above the ECOFFs for these antimicrobials. Hence, E. coli colonies on plates with nalidixic acid were considered QREC and colonies on plates with tetracycline were considered TREC. Plates were incubated for 18 to 24 h at 42 °C and dark green to light blue–green colonies were counted. The within-sample prevalence of QREC and TREC in a sample was obtained by dividing the CFU of E. coli on plates with antimicrobials by the CFU on plates without antimicrobials.The CFU of QREC was determined as follows. A stock solution of nalidixic acid sodium salt at 672 µg/mL  was prepared. Fifty microliters stock solution was added to 1000 µL of the non-diluted and the 10E. coli was selected from a SEC plate without antimicrobial and after subculture susceptibility tested by microdilution according to the recommendations of the Clinical and Laboratory Standards Institute [E. coli ATCC 25922, was conducted in parallel with each batch of isolates tested; all results were within acceptable ranges. ECOFFs , entrance and exit dates as well as reason for exit for all animals present at any time during the study period were obtained from SOMRS. Data used were from the period between the first and the third milk sampling, irrespective of individual birth dates, reproductive cycles, or lactation stages. Reproductive failure and mortality data were defined and prepared according to Beaudeau et al. . The levData on morbidity and antimicrobial treatment were obtained from the farms via repeated questionnaires. At eight occasions during the 2-year study period, from November 2015 to October 2017, farmers/farm staff responded to questionnaires on the health status of the herd. Each questionnaire covered the preceding two months, but for practical reasons, the summer months (May–August) were not covered. Thus, information from each herd was gathered for 16 months of the 24-month study period. BRSV and BCoV infections are less common in the summer months and this period was excluded to ease the workload for the farmers/farm staff. The questionnaire contained questions on the incidence of diarrhea and cough, in calves under the age of six months, in young stock from six months to calving/slaughter (including bulls if present), and cows (lactating and dry). The incidence was rated as “none”, “a few”, “one fourth of the age group”, “half of the age group”, and “more than half of the age group”. For statistical analysis, these questions were transformed to binary variables, such as no diarrhea/cough versus diarrhea/cough in a few or more animals. The questionnaire also contained questions on the number of cows that had experienced the following disease events: mastitis/high SCC, hoof or leg disorders, feed-related disorders (ketosis or inappetence), metritis/retained fetal membranes, fever without obvious reason, and abortion (gestation length < 260 days). Additionally, questions on the number of young stock with hoof or leg disorders or with dullness without obvious reason were included, and on the number of calves up to six months with hoof or leg disorders, umbilical infections, or dullness without obvious reason. Finally, questions regarding the number of lactating cows, dry cows, young stock and calves that had been treated with antimicrobials, irrespective of type, during the period of concern was included. The questions regarding dullness, hoof or leg disorders and umbilical infections in calves had few non-zero variables and were therefore merged into one outcome variable—other diseases in calves. In May each year, a question on vaccination against BRSV or BCoV in the preceding year was added because vaccination could interfere with the result of the antibody testing. After the first postal questionnaire, farmers/farm staff could choose to receive and respond to subsequent questionnaires by post, via the web-based platform Easyresearch , or by telephone. The number of disease events and antimicrobial treatments were adjusted to the mean number of animals in the specific category present in the herd.The effects of BRSV and BCoV were evaluated separately, but it was assumed that all outcomes may be affected by both infections. Therefore, both BRSV and BCoV statuses were forced into all models and the estimates of the effect of one infection were adjusted for the possible effect of the other. Due to the time of infection, all observations within the herd-specific study period were considered as potentially affected by virus infection. Three herds vaccinated against BCoV and one against BRSV and were included as a separate group . Different statistical models were used for different outcomes and each outcome was adjusted for the potential effects of other important covariates based on earlier studies or on biological assumptions, as shown in Where necessary, to meet the assumptions of normally distributed residuals, continuous variables were transformed . DependiSustaining a free status for BRSV and BCoV is beneficial for the production and health of dairy farms, and is possibly also a way to reduce the need for antimicrobial treatments. Measures to uphold a free status likely also reduce the spread of other infectious diseases and antimicrobial-resistant bacteria. In a wider perspective, improved animal health in cattle would reduce the incidence of antimicrobial treatments and thereby the selection pressure towards resistant bacteria.