The structure of multi-domain micromagnetic states in hcp cobalt nanoparticles of spheroidal shape has been studied using numerical simulation in the range of diameters 20–200 nm. The single-domain diameters of the particles are determined depending on their aspect ratio. The complicated vortex structure of domain walls for two- and three-domain micromagnetic configurations is investigated. It has been shown that three domain states are actually strongly deformed two vortex states. In hcp cobalt particles of sufficiently large sizes two types of three-domain micromagnetic states with close total energies have been obtained. They differ in different magnetization directions of the exchange cores of the vortex domain walls. The remanent magnetization of particles has been calculated for two- and three-domain micromagnetic states. The single-domain diameters of fcc cobalt nanoparticles with cubic type of magnetic anisotropy were also calculated. They were also the first magnetic nanoparticles extensively studied theoretically5. However, the theory of small magnetic particles developed by Kittel2, Stoner and Wohlfarth3, Brown4, and Aharoni5 is only capable to describe the magnetic properties of single-domain nanoparticles. Meanwhile, magnetic nanoparticles of submicron sizes are often found in practice. For example, the assemblies of magnetic nanoparticles with a wide size distribution are frequently obtained in chemical synthesis6, so that the sizes of some particles may exceed the corresponding single domain diameter. Particles of sufficiently large sizes can arise during prolonged annealing of thin-film samples7, they can be created as a result of implantation process8, etc. It is worth noting that the magnetic properties of submicron-sized nanoparticles play an important role in paleo- magnetism9.Cobalt nanoparticles attract substantial interest due to their technological significance5. However, the characteristics of nanoparticles of sufficiently large sizes are still poorly investigated. In particular, the dependence of the single-domain diameter of a spheroidal cobalt nanoparticle on its aspect ratio is unknown. The upper and lower analytical estimates for the single-domain diameter of magnetic nanoparticle given by Brown10 are not always close enough. Therefore, in most cases a detailed numerical simulation is necessary to determine the single-domain diameter of particles of various magnetic materials.The magnetic properties of nanoparticles in inhomogeneous micromagnetic states differ significantly from those of single-domain ones12. In addition, modern electronic holography technique17 allows to study experimentally inhomogeneous micromagnetic distributions in magnetic nanoparticles and nanowires. This revives interest in detailed micromagnetic calculations of inhomogeneous magnetization distributions in nanoparticles of submicron sizes. Some practical applications of submicron nanoparticles are also probable. For example, it has recently been suggested18 to use nanoparticles in vortex micromagnetic states in magnetic nanoparticle hyperthermia.At present, domain structures in submicron cobalt nanoparticles and cobalt nanowires are experimentally studied using magnetic force microscopy2 many years ago. Then, Stapper19 computed a one-dimensional magnetization distribution in a cobalt sphere with uniaxial anisotropy subdividing the particle into a large number of thin parallel slices. He showed that the energy of the two-domain state became lower than that of the uniform magnetization if the diameter of the sphere exceeded a critical value, Dс0 = 76.0 нм. Aharoni and Jakubovics20 carried out the two-dimensional calculation of the magnetization distribution in a cobalt sphere under the restriction of cylindrical symmetry. For spherical cobalt particle they found that the cylindrically symmetric magnetization distribution with two domains separated by the cylindrical domain wall had lower energy than the one of Stapper. The single domain diameter of hcp cobalt nanoparticle was determined20 to be Dс0 = 68.2 нм. Similar calculations were carried out also21 for a nanoparticle with cubic magnetic anisotropy.The first simple variational estimate of the single domain diameter for a spherical cobalt nanoparticle was given by Kittel24. Using the 3D numerical scheme the vortex magnetization distribution was first constructed in a magnetically soft cylindrical nanoparticle25. A characteristic feature of vortex distribution is the so called “exchange magnetization core”, i.e. relatively small region near the vortex center where the unit magnetization vector rotates rapidly to the direction perpendicular to the overall vortex magnetization outside the vortex center. This behavior of the unit magnetization vector excludes the magnetization singularity in the vortex center. Later it was realized that the vortex distributions are typical inhomogeneous micromagnetic states in magnetically soft nanoparticles of a rather arbitrary shape, such as a cube28, flat tablets34, spheres and ellipsoids38. It is now well known that the vortex magnetization distribution has as a rule the lowest total energy both in soft magnetic nanoparticles with magnetic anisotropy constant K small compared to the square of the saturation magnetization Ms, K ≪ Ms2, as well as for particles with moderate anisotropy constant value, K ~ Ms2, such as hcp cobalt.However, both one- and two-dimensional micromagnetic calculations significantly limit the possible types of inhomogeneous micromagnetic distributions admissible in a three-dimensional magnetic nanoparticle. Significant progress in numerical modeling of inhomogeneous micromagnetic states was achieved after creating a numerical scheme for three-dimensional micromagnetic calculations38. The complex vortex structure of domain walls separating two and three domain states is revealed. It is shown that in hcp cobalt nanoparticles there are two types of three domain states with close total energy. They differ in the direction of magnetization of the exchange cores of the vortex domain walls. The areas of existence of two and three domain states in hcp cobalt nanoparticles are determined, and the remanent magnetizations of particles in these states are obtained.In this paper the two and three domain states in cobalt hcp nanoparticles are studied in detail using a numerical solution of the dynamic Landau – Lifshitz – Gilbert equation39 in experimentally studied cobalt nanoparticle assemblies.For completeness, inhomogeneous magnetization distributions in spheroidal cobalt fcc nanoparticles with cubic type of magnetic crystallographic anisotropy are also studied. These nanoparticles are often presentMs = 1400 emu/cm3, uniaxial magnetic anisotropy constant K = 4.3 × 106 erg/cm3, exchange constant C = 2 A = 2.6 × 10−6 erg/cm41. Stationary magnetization distributions in spheroidal nanoparticles with transverse and longitudinal diameters D and Dz, respectively, were obtained by solving the dynamic Landau – Lifshitz – Gilbert (LLG) equation. The easy axis of magnetic anisotropy is assumed to be parallel to the particle axis of symmetry, which is directed along the Z axis of Cartesian coordinates. The magnetization distribution in cobalt nanoparticles is approximated by small cubic elements with a size b = 1–2 nm, which is much shorter than the exchange length in this material, 5. The inhomogeneous micromagnetic states are studied for cobalt nanoparticles with aspect ratios 0.5 ≤ Dz/D ≤ 2.0 in the range of transverse particle diameters 20 ≤ D ≤ 200 nm.The following magnetic parameters were used in the numerical simulation of the magnetization distributions in hcp cobalt nanoparticles with a uniaxial type of magnetic anisotropy: saturation magnetization η = Dz/D, near the particle single domain diameter, where a uniform magnetization competes in energy with the transverse vortex state. The axis of the transverse vortex points perpendicular to the particle easy anisotropy axis. It is well known5 that the total energy of uniform magnetization in a particle of an ideal ellipsoidal shape does not depend on the particle size. On the other hand, as Fig. Fig. 1a shows the energy diagram of stationary micromagnetic states in cobalt hcp nanoparticles with various aspect ratios, Dс0. According to Brown’s definition10, the single domain diameter of magnetic nanoparticle, Dc0, is a size below which the uniform magnetization has the lowest total energy among all other stable magnetization distributions. For example, as Fig. η = 1, the transverse vortex remains stable in some range of diameters D < Dc0 = 45 nm. However its total energy in this region is higher than that of uniform magnetization. Therefore, the transverse vortex is metastable state at D < Dc0. Similarly, uniform magnetization remains stable in some range of diameters D > Dc0, where its total energy is higher than that of transverse vortex. Thus, uniform magnetization is metastable state at D > Dc0.The intersection of the corresponding energy curves in Fig. Dс0 = 39.7 nm to Dс0 = 74.2 nm, when particle aspect ratio changes in the range η = 0.5–2.0. Interestingly, with increasing particle size the transverse vortex state is strongly deformed and resembles the two-domain state predicted by Stapper19. However, 3D numerical simulation shows that unlike Stapper’s one-dimensional calculations the domain wall separating nearly oppositely magnetized domains retains a complex vortex structure. As an example, Fig. D = 92 nm and Dz = 64 nm, respectively. In this particular case the exchange core of the vortex is magnetized in the positive direction of the X axis. Figure αx, αy, and αz depending on the coordinate y, 0 ≤ y ≤ D, along the central particle diameter, x = D/2, z = Dz/2. As can be seen in Fig. αz component of the unit magnetization vector takes values close to ±1 in the left and right half of the particle, respectively. The αz component changes sign within the exchange core of the vortex, y ≈ D/2, where αx component approaches the value αx ≈ 1. The αy component is close to zero everywhere along the line considered.According to Fig. x = D/2, z = Dz/4. In this case the distribution of magnetization is more complex. But as before, the αz component is large and positive within the left domain, and negative in the right domain. Thus, in the bulk of the particle the unit magnetization vector is approximately parallel to the easy anisotropy axis direction. This explains why the axis of the vortex in hcp cobalt nanoparticle lays perpendicular to the easy anisotropy axis. If the vortex core were magnetized along the easy anisotropy axis, the magnetization in the main particle volume, outside the exchange core, would be directed perpendicular to the easy anisotropy axis. The magnetic anisotropy energy for such magnetization distribution would be high making this state energetically unfavorable.The distribution of magnetization along the central particle diameter, perpendicular to the axis of the vortex, is used in this work to determine the characteristic width of the vortex domain wall. The domain wall width is determined in a usual manner1, as the thickness of a wall with a constant value of the derivative dαz/dy equal to that at the center of the wall. The characteristic width of the domain wall for particles with different aspect ratios is shown in Fig. In Fig. 19 are currently of historical interest only. Stapper’s hypothesis about the existence of a flat domain wall separating uniformly magnetized domains turned out to be unrealistic. In a cobalt hcp nanoparticle the flat domain wall is energetically unfavorable; the true domain wall has vortex structure. 1D calculations also lead to a significantly overestimated single-domain diameter of spherical cobalt nanoparticle. According to Stapper’s calculations it was determined to be Dс0 = 76.0 nm. However, as Fig. Dс0 = 43.6 nm.The 1D calculations performed by Stapper for cobalt hcp nanoparticleD ≤ 200 nm. Here two types of three-domain magnetization distributions that arise from strongly deformed two vortex micromagnetic states were discovered. For the first type of the three-domain state the exchange cores of vortices are magnetized identically, whereas for the second type they magnetized in opposite directions. Fig. η = 0.5 and 0.7 in the range of transverse diameters 20 ≤ D ≤ 200 nm. According to Fig. η = 0.5 and 0.7 are given by Dc0 = 40, and 41 nm, respectively. Further, three domain configuration becomes energetically favorable with respect to two domain one for the transverse particle diameter D > Dc2, where Dc2 = 120 nm for η = 0.5 and Dc2 = 172 nm for η = 0.7, respectively. On the other hand, the three domain states are stable in the range of diameters D > 88 nm for η = 0.5, and in the range D > 116 nm for particles with aspect ratio η = 0.7, respectively.The multi-domain micromagnetic configurations appear in cobalt hcp nanoparticles of sufficiently large sizes. These states determine the magnetic properties of submicron-sized cobalt nanoparticles and deserve a detailed study. Since reliable numerical simulation of the magnetization distributions in large nanoparticles requires significant computer resources, the calculations of multi- domain micromagnetic configurations in cobalt hcp nanoparticles are performed in this work in a limited range of transverse particle diameters, αx component of the unit magnetization vector obtained numerically in the corresponding diametral sections of the nanoparticle. As can be seen in Fig. The structure of three domain micromagnetic configurations in nanoparticles with a sufficiently large transverse diameter is shown schematically in the upper panels of Fig. D = 200 nm, Dz = 140 nm for three domain state with exchange cores of vortex domain walls magnetized along the positive direction of the X axis. Figure y, 0 ≤ y ≤ D, along the central particle diameter, x = D/2, z = Dz/2. In Fig. y coordinate along the line x = D/2, z = 3Dz/4. The vortex structure of the domain walls separating oppositely magnetized domains is clearly visible in Fig. Figure αx = 1, whereas the other components are close to zero. At the same time, outside the domain boundaries the component αz takes values close to −1 for the side domains and close to +1 for the central domain, while the other components are small. Consequently, in the bulk of the cobalt nanoparticle, the unit magnetization vector is approximately parallel to the direction of the easy anisotropy axis. This leads to a significant decrease in the magnetic anisotropy energy of the particle. As Fig. x = D/2, z = 3Dz/4, a non-zero αy component of the unit magnetization vector appears, which has different signs in the left and right half of the nanoparticle. The appearance of the αy component outside the diametral plane is associated with the vortex character of the domain walls.As Fig. αx = 1, whereas in the center of the right domain wall αx = −1. Otherwise, the magnetization distributions for both three domain states are very close.Figure 43 that in addition to hcp cobalt nanoparticles, cobalt nanoparticles with fcc crystal structure can also be found in the experiment. For completeness, we calculate single-domain diameters for cobalt nanoparticles with fcc crystal structure having cubic type of magnetic anisotropy. In the calculations the cubic anisotropy constant was taken to be Kc = 2.7 × 106 erg/cm342, the particle saturation magnetization and the exchange constant being the same as for cobalt hcp nanoparticle.Ii is well knownDc0, shown in Fig. Dz/D < 1, the axis of the vortex is parallel to the particle axis of symmetry (Z axis). However, for elongated cobalt fcc nanoparticles, Dz/D > 1, the transverse vortex is energetically favorable.Figure 20. These calculations are of fundamental importance for Micromagnetics5 since such important quantity as the nanoparticle single-domain diameter is determined10 from a comparison of the total energy of a uniform magnetization with the energy of the lowest inhomogeneous micromagnetic state. In the pioneering work of Stapper19 the structure of a flat Bloch domain wall in a spherical cobalt nanoparticle with uniaxial magnetic anisotropy was calculated. The two domain state of Stapper is shown schematically in Fig. Dс0 = 76.0 nm.Numerical calculations of inhomogeneous magnetization distributions in spherical and ellipsoidal magnetic nanoparticles have a rather long history20 the existence of plane domain structures constructed by Stapper, since the two-domain and three-domain micromagnetic states of Stapper break the initial axial symmetry of a spherical nanoparticle with uniaxial magnetic anisotropy. They proposed21 a 2D scheme of micromagnetic calculation, in which the distribution of magnetization in a spherical nanoparticle was approximated by small numerical cells of a toroidal shape. As the lowest energy inhomogeneous micromagnetic state they considered the structure of an axially symmetric cylindrical magnetic domain, shown schematically in Fig. 20 found this structure to be energetically favorable in comparison with the two domain state of Stapper, Fig. Dс0 = 68.2 nm for the single-domain diameter of the hcp cobalt spherical nanoparticle, smaller than that of Stapper19.However, Aharoni and Jakubovics questioned19 and 2D [20.21] micromagnetic calculations significantly limit the possible types of inhomogeneous micromagnetic distributions allowable in three-dimensional magnetic nanoparticle. In fact, it can be shown that trial magnetization distributions shown in Fig. 37 showed that in magnetic nanoparticles with a small and moderate value of the anisotropy constant the vortex is the lowest energy inhomogeneous micromagnetic state. In a magnetically soft spherical nanoparticle, K ≪ Ms2, with a diameter slightly exceeding the single domain one, the vortex arises as a result of instability of a uniformly magnetized state. As a result, the axis of the vortex is parallel to the easy anisotropy axis. This configuration can be stable in a certain range of diameters near the single domain diameter38.Unfortunately, both 1DK ~ Ms2, only the transverse vortex state is stable. As shown in this paper, this is true, in particular, for cobalt hcp nanoparticles. The transverse vortex state in hcp cobalt nanoparticle is shown in Fig. Dс0 = 43.6 nm, that is, much smaller than that for cylindrical magnetic domain20.However, with increase in the particle diameter the reduced radius of the exchange vortex core decreases and the rotation of the vortex axis into a plane perpendicular to the easy anisotropy axis turns out to be energetically favorable. As noted above, this process leads to a decrease in the particle magnetic anisotropy energy. On the other hand, for particles with a moderate value of the uniaxial anisotropy constant, In this paper, inhomogeneous magnetization distributions in spheroidal cobalt nanoparticles are obtained in a wide range of particle sizes based on the solution of dynamic Landau – Lifshitz – Gilbert equation. The complex vortex structure of domain walls separating two and three domain micromagnetic configurations is studied. It is shown that in cobalt hcp particles there are two types of three domain states with close total energy but different directions of magnetization of the exchange cores of the vortex domain walls. The domain wall width is calculated as a function of the transverse particle diameter. The areas of existence of three domain states in cobalt hcp nanoparticles with different aspect ratios are obtained. The remanent magnetizations of cobalt nanoparticles in multi-domain states are calculated.For completeness, single-domain diameters are also calculated for cobalt fcc nanoparticles with cubic type of magnetic crystallographic anisotropy. It is shown that the single domain diameter for cobalt hcp nanoparticle significantly exceeds the corresponding value for cobalt fcc particle with the same aspect ratio. It seems that the numerical results obtained will be useful in interpreting experimentally observed domain structures in submicron-sized magnetic cobalt nanoparticles.36γ is the gyromagnetic ratio and κ is the phenomenological damping constant. The effective magnetic field 36V is the nanoparticle volume, Ms is the saturation magnetization, C is the exchange constant, and 36K and Kc are the uniaxil and cubic anisotropy constants, respectively.Dynamics of the unit magnetization vector b much smaller than the exchange length N ~ 103–104, is necessary to approximate with sufficient accuracy the vortex type magnetization distribution in nanoparticle volume. In the present calculations the magnetic damping parameter is given by κ = 0.5.For numerical simulation a non single-domain nanoparticle is approximated by a set of small ferromagnetic cubes of side i-th numerical cell, respectively.To find all stable micromagnetic states existing in a nanoparticle with given sizes and magnetic parameters it is necessary to carry our different runs of numerical simulations starting from various initial magnetization distributions. To save the time of numerical simulation in some cases one can use initial trial magnetization distributions resemble those expected for final stable micromagnetic configurations. In accordance with the Eq. , the fin


Stabilin-2/HARE is the primary clearance receptor for circulating hyaluronan (HA), a polysaccharide found in the extracellular matrix (ECM) of metazoans. HA has many biological functions including joint lubrication, ocular turgor pressure, skin elasticity and hydration, cell motility, and intercellular signaling, among many others. The regulatory system for HA content in the tissues, lymphatics, and circulatory systems is due, in part, to Stabilin-2/HARE. The activity of this receptor was discovered about 40 years ago (early 1980s), cloned in the mid-1990s, and has been characterized since then. Here, we discuss the overall domain organization of this receptor and how it correlates to ligand binding, cellular signaling, and its role in known physiological disorders such as cancer. The focus of this review is the interactions of hyaluronan (HA) with Stabilin-2 in both physiologically normal and diseased states. HA is a long unmodified polysaccharide containing alternating sugars consisting of glucuronic acid and N-acetylglucosamine. Stabilin-2 is one of two members of the scavenger receptor class H family and is the only one that binds HA. Although fifty-five percent of the amino acids that compose Stabilin-1 are homologous to those of Stabilin-2, it does not bind HA, and thus Stabilin-1 will not be reviewed in this manuscript. The biological activity of Stabilin-2 was first discovered by Fraser et al. in experiments in which they were calculating the clearance of HA in the blood of rabbits in the early 1980s ,4. The p2+, Mg2+ or Ca2+ [Fas1: Recombinant cells that express high levels of Stabilin-2 often become sticky in that they stick to each other. Park et al. investigated how this occurred and found that Fas1 domains are responsible for homophilic cell–cell adhesion which also required a divalent cation such as Mn or Ca2+ . Fas1 do or Ca2+ . Further or Ca2+ . Since S2+ and occurred at pH 7.3, but was optimal at pH 6.8. Furthermore, the intriguing part of this story is that the second EGF domain in each cluster is more atypical than the other EGF domains and there is a conserved histidine which aligns with position 1403 of the third cluster. This histidine is conserved within each EGF cluster and between both Stabilin receptors. Mutagenesis of the histidine did not affect binding affinity for PS, but did affect binding enhancement at lower pH, suggesting that the histidine is protonated at lower pH (pKa ~6.0) and that other residues in the binding loop are involved with PS interaction [EGF: The inner leaflet of the plasma membrane of erythrocytes is enriched in phosphatidylserine (PS), which flips to the outer leaflet when the cells die . The experaction .Link: The Link domain is the HA binding domain for Stabilin-2. It is located between the last EGF and Fas1 domains near the transmembrane region. Deletion of the Link domain results in complete ablation of HA binding ,22. The As of this year (2020), there are 20 known ligands of Stabilin-2 and, despite this diversity, the protein receptor remains a mundane sequence of EGF and fasciclin domains. Stabilin-2 is composed of 2551 amino acids, of which 2458 are part of the extracellular domain, 21 are the single transmembrane domain, and the remaining 72 amino acids are the intracellular domain. There are 21 epidermal growth factor (EGF) and EGF-like domains in four clusters containing five to six domains per cluster . Each EG35S pulse-chase experiments, we know that HARE expression in Stabilin-2 expressing cells begins after the receptor is fully glycosylated; therefore, we have speculated that the cleavage event occurs somewhere between the trans-Golgi network and the cell surface [As alluded to previously, HARE is a proteolytically processed form of Stabilin-2 in which the N-terminal 1135 amino acids are cleaved from the remaining 1416 amino acid receptor bound to the membrane at serine 1136. Recombinant cells expressing only HARE show great ligand binding and endocytic activity. Recombinant cells expressing the full-length Stabilin-2 cDNA express both Stabilin-2 and HARE in which the ratio of the two receptors is about 8-10:1 . From 35 surface . We have surface . Secrete surface ,30. The  surface . ReducinThe 93 amino acid Link domain of Stabilin-2 is 65% homologous and 46% identical with the Link domain of Stabilin-1. The cysteine residues are perfectly aligned, but the Stabilin-1 Link lacks the key lysine, tyrosine and additional residues that are predicted for HA binding based on the TSG-6 model ,31 Figu. Though d value is 7.2 ± 1.2 nM and that there are about 118,000 binding sites or receptors on the cell surface [Similar experiments were repeated for hHARE after it was stably expressed in Flp-In 293 cells. The chondroitin sulfate species with the highest to lowest apparent affinities in the endocytosis competition assay were: CS-A, , and DS. Once again heparin, heparan sulfate, keratan sulfate did not compete. None of these GAGs competed for HA at 4 °C. Scatchard analysis of HA binding in hHARE cells reveals that there is a single class of non-interacting binding, suggesting that there is one receptor in these recombinant cells that binds HA. This analysis also revealed that the affinity or KWith the development of using purified protein and individually labeled ligands, it was possible to screen without competing a known ligand such as HA. This overcomes the weaknesses of the competition assay such as (1) a weak ligand may not sufficiently compete with a strong ligand, (2) protein conformational change for one ligand may prevent the other ligand from binding, and (3) other non-competing binding sites will not be detected. To address this, a secreted ecto-domain of both human Stabilin-2 isoforms (s315 for the large Stabilin-2 receptor) and sHARE  were created. Through a series of assays including ligand blots and an in-house development of an ELISA-like assay, the Link domain of sHARE binds HA, which was competed with by CS-A, CS-C, and CS-D. Both CS-E and DS bind to two sites and do compete slightly with HA bound in the Link domain. However, heparin binding was entirely independent of HA binding. This also confirmed that heparin does not bind within the Link domain and both ligands may bind sHARE simultaneously. CS-E and DS also compete with heparin at the second site outside of the Link domain .2497, Thr2523, Ser2537, Tyr2519, and Tyr2531. Of these, Tyr2519 was confirmed to be phosphorylated through a variety of biochemical and molecular approaches and interacts with the mitogen-activated protein kinase pathway (MAPK), specifically the extracellular signal-regulated kinases (ERK1/2) and not JNK or p38. The Ras-Raf-MEK-ERK cascade, or MAPK pathway, is involved with numerous cellular responses such as transcription, cell survival, migration, cytoskeletal remodeling, differentiation, and cell cycle progression in proliferation [The cytoplasmic domain contains four Tyr, seven Ser, one His and five Thr amino acids, which are all potentially phosphorylated . Using Nferation . This paferation . This su2519 for alanine also significantly affected endocytosis, suggesting that receptor signaling upon ligand binding is a coordinated event [The cytoplasmic domains of both Stabilin receptors are very diverse, without any homology. As seen in ed event . We nexted event . Since bed event . The cured event ,38. Thised event . Using ted event . Why woued event . HA in ted event .2280 which is situated in the Link domain [2280 contained N-glycans that were sialylated. Mutagenesis of this Asp to Ala did not affect the endocytosis of HA, but did affect binding in some in vitro assays and we think that the absence of the glycan destabilized either the binding of HA with the protein or slightly altered the topology of the Link domain which lowered affinity in an in vitro experimental setting. With regard to signaling, the ablation of the glycan at position 2280 resulted in HA binding with the receptor without activation of NF-kB. In contrast, the N2280A mutant did not affect NF-kB signaling by heparin, DS, or AcLDL [In an extension to the investigations of which ligands bound to HARE , we askeor AcLDL  suggestiStabilin-2 is expressed in the endothelium of liver , lymph nBiomolecules [Recently, it was discovered in specialized structures of the muscle . In the olecules .Both Stabilins are expressed in bone marrow and are involved with the clearance of HA and other extracellular matrix molecules in that localized area. Within the marrow, the endothelia is a migration site for hematopoietic stem and progenitor cells (HSPCs) between the blood and bone . Do the www.proteinatlas.org) and other similar databases using RNA expression data show that Stabilin-2 expression is highest in the spleen. A key word search in PubMed using “Stabilin-2 and spleen” brings up 11 references, none of which are a detailed study of Stabilin-2 function in spleen. A similar search in the Web of Science database by Clarivate Analytics produces similar results. What is the function of Stabilin-2 in spleen? We assume that it may be similar to bone marrow in that there is local clearance by the extracellular matrix and other functions that are specialized in the spleen, such as the enhanced elimination of dead blood cells, and, possibly, elimination of bacteria [The human protein atlas  will find a home in certain compatible tissues (soil) to continue growing in a suitable environment . TissuesThe lymphatic system or “second circulatory system” is a common transportation highway for metastases and tumor lymphangiogenesis and is common among the various types of cancer. The lymph node itself highly expresses Stabilin-2 and acts as one of the entrapping receptors for metastatic tumor cells. A study in which the investigators injected PC3M-LN4 prostate cancer cells that are known to be rich in pericellular HA in mice, found that when Stabilin-2 was blocked with a blocking antibody prior to tumor injection, the number of lymph node metastases was dramatically reduced . A folloStabilin-2 is the primary scavenger receptor for HA as mentioned previously. Mice lacking Stabilin-2 expression (stab2KO) have very high HA levels in their circulating plasma, though the mice are physiologically normal in all other aspects . The rolCD44 and its splice variants are enriched in a variety of cancer cells and all of these receptors bind HA ,78. TherBy definition, Stabilin-2/HARE is an excellent scavenger receptor for the following reasons: (1) it binds and internalizes both natural and synthetic ligands, (2) it continuously recycles from the cell surface to endosomes to accumulate exogenous cargo, and (3) the expression is relatively high in specific cell types of tissues and organs. Moreover, the function of Stabilin-2/HARE is ever expanding to include aspects of development. The recent discovery that Stabilin-2 is involved with myotube development was briefly covered . There aE. coli to express his constructs in Park et al. [E. coli K12 Shuffle T7 Express Stain to express the seventh Fas-1 domain for crystallization, though this is one of the few areas of the protein without any cysteines [One of the big impediments of characterizing this receptor on the atomic level is the lack of a known structure. Both isoforms contain an abundance of cysteine amino acids which are difficult to fold in bacterial-based expression systems. Dr. In-San Kim’s lab used an Origami strain of k et al.  and corrysteines . Their rThe hepatic and renal systems eliminate nearly all of the foreign molecules that are either injected or consumed. Very small molecules tend to be eliminated through the kidney, whereas larger molecules or more complex structures such as polymers or nanoparticles are cleared through the liver ,89. With


The acyl carrier protein (ACP) domain shuttles substrates and reaction intermediates in type I fungal fatty acid synthases via transient protein-protein interactions. Here, using electron cryo-microscopy (cryoEM), we report the structure of a fungal FAS stalled at the dehydration reaction, which precedes the final enoyl reduction in the fatty acid biosynthesis cycle. This conformation revealed multiple contact sites between ACP and the dehydratase (DH) and enoyl reductase (ER) domains that occluded the ACP binding to the adjacent ER domain. Our data suggests a minimal path from the DH to the ER reaction site that requires minute changes in the coordinates of the structured N- and C- termini of the ACP domain. Lou and Mazhab-Jafari report the structure of a fungal fatty acid synthase stalled at the dehydration reaction, which precedes the final enoyl reduction in the fatty acid biosynthesis cycle. This study suggests that the binding of acyl carrier protein domain to its proximal dehydratase and enoyl reductase domains in fatty acid synthase is mutually exclusive. Therefore, de novo fatty acid synthesis in human and pathogenic species  and malonyl-CoA substrates using NADPH as a reducing agent.Lipids constitute one of the major building blocks of life and define the boundaries of cells. They play key roles as signaling molecules and deregulation of lipid metabolism forms the basis of many disorders including type 2 diabetes, cardiovascular diseases, cancer, and infectionsies Fig.  is an at5 and transient protein–protein interactions are governed by random diffusion. On the other hand, type I FAS, found in actinobacteria and eukaryotes, is an ‘associated system’ where all essential enzymes are expressed by one or two genes and organized into a large, multi-functional assembly line6. Substrate and reaction intermediates are shuttled between catalytic centers via a flexibly tethered acyl carrier protein (ACP) domain.Based on the organization of catalytic domains, two FAS types have been defined. Type II FAS, found in bacteria and plants, is a ‘dissociated system’ where each catalytic enzyme is expressed via a unique gene. Each enzyme works as a monofunctional unit10. In fungi, one or two genes encode all the essential catalytic domains and additional segments that serve as structural domains, stabilizing the assembly into a barrel shaped D3 symmetric dodecamer of ~2.6 megadalton in size. The assembly is composed of two C3 symmetric reaction chambers harboring three identical sets of catalytic domains  domain and at a lower signal-to-noise ratio (S/N) compared to other regions of the protein8. Low resolution (~20 Å) densities with weak S/N are also observed in proximity of other catalytic centers in a cryoEM map of S. cerevisiae FAS12 that allowed for approximate placement of ACP models in proximity of KS, ketoacyl reductase (KR), ER, and acetyltransferase (AT) domains albeit with ambiguity in ACP orientation. Recent cryoEM studies have observed sub-nanometer resolution ACP densities proximal to KS14, ER15, and AT16 domains in the apo state of FAS enzymes purified from different fungal species.In the crystal structures of FAS from 9. CryoEM studies of endogenous FAS from S. cerevisiae and Candida albicans14 have shown that stalling catalysis at the KS domain using cerulenin, a covalent KS inhibitor, resulted in translocation of ACP toward the inhibited domain in the presence of substrates. Therefore, we asked if it is possible to apply this strategy to other catalytic sites of FAS and image ACP at different locations within the reaction chamber. In this study, we prepared a recombinant FAS from the model organism S. cerevisiae allowing mutagenesis for stepwise inactivation of each catalytic site.The mechanism of ACP-mediated substrate shuttling in fungal FAS is not completely understood. Surface electrostatic complementarity has been suggested as a contributing factorS. cerevisiae and Rhodosporidium toruloides can be purified by heterologous expression in bacteria with the purified complex retaining catalytic activity19. These observations strongly suggest that the FAS mega-enzymes undergo self assembly. We therefore cloned FAS1 and FAS2 genes from S. cerevisiae genomic DNA into pET28 and pET15 expression vectors, respectively, and expressed and purified the recombinant FAS from E. coli BL21 strain with a 6xHis tag on the C terminus of FAS1 polypeptide  and therefore report on the averaged ACP densities at each catalytic site.To enable site-specific inactivation of each catalytic site, we sought to prepare a recombinant FAS to facilitate mutagenesis. Recent studies have shown that the multi-megadalton fungal FAS from S. cerevisiae FAS as a guide7, we systematically mutated key residues involved in catalysis to alanine in the AT (S274A), MPT (S1808A), KR (Y839A), DH (H1564A), and ER (H740A) domains  the docked ACP domain, (ii) two sub-domains of DH, and (iii) part of ER domain that contacted the ACP stalled at the DH domain. This ER fragment also encompassed an ACP binding site that was observed in previous studies of fungal FAS, where the shuttling domain was seen adjacent to the catalytic cavity of the ER domain21 for bacterial type II FAS  with multiple elements in the β-chain. The structural domain of the ACP has so far only been observed to contact the structural insertion of the KS domain that forms part of the central α-disc of the FAS barrel8. The interaction of ACP with the ER15 and AT16 domains is shown to be mediated by its catalytic lobe, with no contacts between the structural lobe of the shuttling domain and the β-chains. The mode of interaction of ACP with ER is likely conserved between different species of fungi, as similar relative orientations have been observed in at least three different species  is weak and transient interactions between the ACP and the interior of the FAS reaction chamber. The localization of the ACP domain that we observed upon stalling catalysis at the dehydratase step provided the opportunity to define the binding site of this shuttling domain at the DH. Both the catalytic and structural lobes of the ACP interact  can satisfy a domain translocation of the ACP from the DH domain to the proximal ER. Therefore, no changes in the ACP N- and C-terminal linker conformation are needed for transition between these successive reactions. This contrasts with an order of magnitude larger displacement of ACP linkers if an ACP docked at a DH domain were to translocate to an ER domain of another β-chain protomer  was obtained by phenol/chloroform extraction. FAS1 and FAS2 genes were amplified from genomic DNA using primers containing overlaps with the vector backbone for cloning with NEBuilder HiFi DNA Assembly (NEB) (Supplementary Table FAS1 was cloned into NcoI-digested pET-28a(+) with a C-terminal His6 tag (JWL02). FAS2 was cloned into NcoI-digested pET-15b(+) (JWL03). The sequences of the FAS1 and FAS2 genes were verified  until the OD600 nm reached 0.6. The culture was cooled to 15 °C and expression was induced with 0.5 mM IPTG. Expression occurred at 15 °C, 180 RPM overnight.The JWL02 and JWL03 plasmids were co-transformed into g for 15 min and resuspended in lysis buffer . Resuspended cells were incubated with DNAse I and lysozyme for 10 min. Cells were lysed by sonication  in an ice-water bath with five cycles of ~15 W pulses every 0.5 s for 1 min followed by 2 min rest. The lysate was cleared by centrifugation at 40,000 × g for 1 h. The cleared lysate was filtered using a 0.22-μm filter and loaded onto a pre-equilibrated 5 mL HisTrap column . The column was washed with 10 column volumes of wash buffer  before elution with a linear gradient of 0–100% elution buffer  over 20 column volumes. Fractions were pooled and concentrated to 1 mL and injected into a Superose6 10/300 GL column  pre-equilibrated with TBS . Gel filtration fractions were pooled and concentrated to 2 mg/mL, flash frozen in liquid nitrogen and stored at −80 °C. For cryoEM samples, proteins were used fresh on the day of purification.Cells were harvested by centrifugation at 4000 × 14. Briefly, 20 μg FAS was mixed with 0.2 mM acetyl-CoA, 0.7 mM NADPH, 1 mM DTT, and 100 mM potassium phosphate pH 7.4 in a 100-μL reaction. The level of NADPH was monitored at 340 nm for 3 min and the reaction was started by adding 30 nM malonyl-CoA and monitored for 1 h. The specific activity of wild type recombinant FAS was measured from three independent protein preparations to be 78 ± 32 mU/mg, where one unit is defined as consumption of 1 μmol of malonyl-CoA per minute. Only linear part of the curve  was used for calculation of the specific activity25.Activity assays of purified recombinant FAS were performed as described26 mounted in a Vitrobot Mark IV. Blotting was done for 3 s at 4 °C and 100% humidity before plunge freezing in liquid ethane. A total of three grids were prepared per condition reported in Supplementary Table To prepare samples for CryoEM imaging, 3 μl of freshly purified protein complexes were added at a concentration of 1–2 mg/ml to glow-discharged (25 s in air) holey gold grids−/Å2/frame (Supplementary Table 27.Electron micrographs were collected with a FEI Tecnai F20 field emission electron microscope equipped with a Gatan K2 summit direct detector device (DDD) camera. Micrographs were acquired as movies in counting mode using DigitalMicrograph® software with 1.45 Å/pixel, 2 frames/s for 15 s, and an exposure rate of 1.2 eStock solutions of acetyl-CoA, malonyl-CoA, and NADPH were prepared at a concentration of 10 mM in TBS buffer. For the AT and MPT double mutant FAS construct (S274A - S1808A), 1 μL of either acetyl-CoA or malonyl-CoA stock solutions were diluted with TBS to 5 μL before addition to 10 μg (at 2 mg/ml) of purified FAS, before application onto cryoEM grids.For KR (Y839A), DH (H1564A), and ER (H740A) mutants, 100 nmol NADPH and 50 nmol malonyl-CoA were added to the pooled and concentrated HisTrap fractions  and incubated for 1 h at room temperature. Substrates were removed by gel filtration as described above. In total, 10 μg (at 2 mg/ml) of purified FAS was mixed with a 5-μL solution containing 10 nmol acetyl-CoA, 25 nmol NADPH, and 15 nmol malonyl-CoA. Samples were kept on ice for 30 min before application onto cryo-EM grids.For the WT FAS, 10 μg of purified FAS at 2 mg/ml was mixed with solutions containing either 5 μL of TBS (for the apo sample), or 5 μL of TBS containing 10 nmol acetyl-CoA, 25 nmol NADPH, and 15 nmol malonyl-CoA (for turn-over conditions) on ice, respectively. Samples were applied onto the cryoEM grids immediately after the addition of TBS plus substrates.28. Movies were aligned by alignframe_lmbfgs implemented in cryoSPARC 2.029. CTF estimation was done with CTFFIND430. Particle motion correction was performed with alignpart_lmbfgs implemented in cryoSPARC 2.029. High resolution refinement was done with the homogenous refinement algorithm using particles selected from reference free 2D classification. A 30-Å low-pass filtered cryoEM map was generated from PDB: 2UV87 with ACP atoms deleted from the model. This map was used as the initial reference in the homogenous refinement. Model to map conversion was carried out in UCSF Chimera31. Low-pass filtering of refined maps was carried out using the ‘relion_image_handler’ command. All one-voxel-thin slices were generated using XIMDISP32.Data presented in Supplementary Table 33. Movies were aligned with the Relion implementation of MotionCor2 and CTF estimation was performed using CTFFIND4. Following 2D classification, all 3D classification was done with C1 symmetry using a 30-Å low-pass filtered cryoEM map generated from PDB 2UV8 with ACP atoms deleted as the initial reference. Particle images were selected from 3D classes and refinement was done with C1 symmetry. The refined maps were aligned to the D3 symmetry axis and a second refinement was carried out with D3 symmetry imposed. The reference was aligned to the symmetry axis using the ‘relion_align_symmetry’ command.Data presented in Supplementary Table S. cerevisiae FAS8. The residues were 601–704  and residues 1236–1658 (DH pseudo-dimer). ACP residues 140–302 from the α-chain of FAS (PDB: 2UV8) were rigid body docked into the D3 refined map of DH-stalled FAS in the asymmetric unit with the docked β-chain fragments. This model comprising partial segments of ER and DH domains plus the whole ACP domain docked at the observed ACP density in the DH-stalled state was used to generate the initial reference and was low-pass filtered to 15 Å for the focused classifications described below. To create a mask that covered the partial model and the position of the ACP in the ER binding mode, we expanded the aforementioned partial model of ER-DH -ACPDH by including a second model of the ACP domain docked at the ER binding site based on homology to previously observed ACP bound to ER in fungal FAS (PDB: 6U5V)14. A 15-Å resolution mask (i.e. focused mask) comprising the residues mentioned above was generated with UCSF chimera and Relion 3.0. The focused mask was extended by 3 pixels (1.45 Å/pixel) with 3 pixel padding. This mask was subtracted from a mask generated from the D3 refined map of DH-stalled FAS to produce a subtracted mask. The subtracted mask was then multiplied by the map and the ‘modified map’ containing all regions of FAS except for the region defined by the focused mask, was used for subsequent steps.A mask for focused classification was made by rigid body docking of a model of a β-chain protomer and the ACP domain from an α-chain protomer from the FAS crystal structure (PDB: 2UV8) to an asymmetric unit of the D3 refined map of the DH-stalled FAS. Residues from the ER and DH domains of the β-chain were selected based on proximity to the observed ACP density in the DH-stalled state and the domain boundaries identified in a previous crystallographic study of 34. Different projections from the ‘modified map’ were then generated based on orientation information from the D3 symmetry refinement. These projections were subtracted from the symmetry expanded particle images. Subtracted images were used for focused 3D classification without orientation search in Relion 3.0 with an optimized regularization parameter of 500. The focused refinement was done in cryoSPARC 2.0 by importing the subtracted particle data set from Relion 3.0 belonging to the ACP containing 3D class in Fig. The rotation matrix for each asymmetric unit of FAS  was computed using the original particle images with alignment information from the D3 symmetry refinement (done in Relion 3.0) via the ‘relion_particle_symmetry_expand’ command7) was used for docking. Maps and models were visualized with UCSF chimera31 and PyMol35. Flexible fitting was done for Fig. Model fitting was done by rigid body docking of the selected regions of ER, DH, and ACP domains, as described in the manuscript text, into the cryoEM maps of the stalled complexes. An atomic model of FAS  were chosen for alignment using BLASTp36. The β-chain sequence of S. cerevisiae FAS was chosen as the search template. A maximum number of 100 sequences was chosen for initial display. Sequences belonging to different strains of S. cerevisiae were excluded from the analysis. The remainder encompassed 58 sequences from other species of Saccharomycotina (Supplementary Data 37. LOGO plots were generated via the WebLogo server38 with the y-axis set to represent frequency of amino acids plotted against amino acid number (based on S. cerevisiae sequence) on the x-axis.Non-redundant protein sequences corresponding to the subphylum Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary


Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.Channelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga  Tashiro et al. describe a new channelrhodopsin variant from a terrestrial algal species and the role of the C-terminal domain in regulatory function. This far-blue-shifted channelrhodopsin may contribute to optogenetic tool research in the future. Channelrhodopsin-1 and -2 from Chlamydomonas reinhardtii (CrChR1 and CrChR2) were the first ChRs to be discovered, characterized, and the latter was extensively utilized for optogenetic applications. These proteins conduct cations such as H+, Na+, K+, and Ca2+. High-resolution X-ray structures of ChRs revealed details of the molecular architecture and provided insight into the photoactivation and ion conduction pathway5. ChRs have been applied to generate action potentials in the light-insensitive cells and tissues with unprecedented spatio-temporal precision, which initiated a new research field, optogenetics7. ChR variants have been engineered to improve the functionality, and homologous ChRs were also reported accordingly8. Anion-conducting ChRs (ACRs) have been created artificially or naturally discovered11. These proteins are applied in neuroscience to optically silence action potential.Channelrhodopsins (ChRs) are directly light-gated ion channels naturally found in the eyespot of green algaeKlebsormidium flaccidum) was sequenced, and analysis of the genome delineated the molecular basis of the adaptation of land plants12. K. flaccidum was renamed as Klebsormidium nitens . K. nitens adapts to diverse environmental conditions such as desiccation, light, and temperature gradients13. However, the sensory photoreceptor landscape of K. nitens has not yet been established. For the first time, we identified a gene encoding a putative channelrhodopsin from K. nitens (KnChR). A unique feature of KnChR is that it includes the FimV domain at the C-terminus of rhodopsin. FimV is a peptidoglycan-binding protein that is responsible for twitching motility in Pseudomonas aeruginosa and other microbes14. All so far known ChRs comprise a few hundreds of amino acid residues in their cytoplasmic domains, for example, full-length CrChR1 and CrChR2 contain 712 and 737 amino acids respectively3 of which the 7-transmembrane (TM) rhodopsin domain is made up of only about 300 amino acids. Thus, 400–450 amino acids are located mainly on the cytoplasmic side. However, no significant homology was found among the long cytoplasmic extension of the characterized ChRs from different organisms. Since the discovery of ChRs, the role of cytoplasmic domains remains elusive. The initial reports of CrChR1 and CrChR2 described that truncation of the cytoplasmic C-terminal domain did not alter the ion channel function3. Thus, researchers investigated their molecular properties by using truncated proteins carrying only the 7-TM domain. However, an extensive study of the phosphoproteome of the eyespot fraction of C. reinhardtii showed three phosphorylation sites for CrChR1 and one for CrChrR215. Thus, a kinase-dependent regulation of function was also proposed. Recently, it has been shown that phosphorylation of CrChR1 regulates photomotility and calcium signaling of green alga16.Recently, the genome of terrestrial green alga .K. nitens revealed existence of several rhodopsin-encoding genes (http://www.plantmorphogenesis.bio.titech.ac.jp/cgibin/blast/blast_www_klebsormidium.cgi). Among them, we found one gene that is highly homologous to chlorophyte cation ChR such as CrChR2. We named it KnChR in this study. KnChR possesses 831 amino acids and harbors a membrane-embedded 7-TM rhodopsin domain (amino acids 1–272) followed by a long C-terminus region (273–831 amino acids)  between 410–690 amino acids  for functional expression in a mammalian system and fused it with the p3.0-eYFP vector with eYFP at the C-terminus of KnChR. The vector contains the signal peptide for export to ER and membrane trafficking to improve membrane localization. After transfection of the fusion expression construct, we observed weak eYFP fluorescence on the cellular membrane, indicating poor expression in ND7/23 cells. A voltage-clamp measurement showed no photocurrent. We anticipated that low expression was due to a long C-terminus domain (~540 amino acids) after the 7-TM domain. Thus, we systematically truncated the C-terminus to various lengths. We created five variants with different lengths, carrying 697, 397, 310, 290, and 272 amino acids. Note that only the variant with 697 amino acids included the FimV domain. In addition, we fused a c-Myc epitope tag at the C-terminus of the 697 amino acids variant to detect immune fluorescence. Figure Mining of the genomic database of We then performed patch-clamp recordings of each variant in detail. All the constructs having various amino acid lengths are depicted in Fig. The photocurrent amplitudes also differed among the nine variants Fig. . The ampThe current-voltage relations (I-V plots) of KnChR and CrChR2 are depicted in Fig. To compare rectification, I-V plots of CrChR2 and KnChR were created after normalizing the current amplitude (at +50 mV as 1.0) . To our knowledge, the λmax of 430~440 nm is one of the shortest among naturally occurring ChRs.The action spectrum, a wavelength dependency of photocurrent amplitude, was measured with a multicolor light source. Figure p (peak component) and Is (steady-state component), grew as light intensity increased. The Is of the 272 variant became saturated at about 0.09 mW/mm2  was determined to be 0.051 mW/mm2 (KnChR-272), 0.712 mW/mm2 (KnChR-397) and 0.731 mW/mm2 (CrChR2). This indicates that the C-terminal length affected the light sensitivity of KnChR.We then measured the light-power dependency of two KnChR variants (272 amino acids and 397 amino acids), comparing them to CrChR2 Fig. . Photocumm2 Fig. . Note thids Fig. . The hal+ was the conducting ion. The I-V plot under several ionic conditions is depicted in Fig. + conductance across KnChR   of KnChR and CrChR2 are depicted in Figs. Erev measured after substituting the test ion for the control ion, but also on the actual concentrations of the two ions. Taking into account ion concentrations used for the measurements, we estimated permeability ratios  was estimated as 12 × 10−7, while that of CrChR2 was 1.1 × 10−7, indicating KnChR is ~10-fold higher permeable for Na+. The permeability ratio of K+ to H+ (PK+/PH+) of KnChR is about 14-fold higher than that of CrChR2 (8.7 × 10−7 and 0.63 × 10−7 respectively). Interestingly, the KnChR permeability ratio of Ca2+ (PCa2+/PNa+) is higher (0.25) than that of CrChR2 (0.15).We predicted that KnChR functions as a cation channelrhodopsin considering its sequence similarity to chlorophyte channelrhodopsins . Ion selectivity was tested by patch-clamp recording. We used the KnChR 272 amino acids variant for all experiments. Sodium in the extracellular solution was systematically replaced with various cations, while the intracellular solution was fixed  Fig. . A similion Fig. . Altogetos Table . KnChR aIp). In addition, the steady-state component (Is) were analyzed to estimate Erev and ΔErev. Essentially the same results were found . KnChR possesses a large cytoplasmic domain made of about 540 amino acids which might involve a peptidoglycan binding moiety (FimV). For the first time, we demonstrated that the C-terminus of KnChR affects the light-gated ion channel function. The channel closure rate (τ-off) slowed down from 10 to 130 ms as the cytoplasmic domain was shortened , the full-length HKR has been shown to exhibit a normal photocycle which reverts to the initial dark state in 20 s, whereas truncation of the enzyme domain at the C-terminus region resulted in the production of a bi-stable photocycle in which rhodopsin possesses two dark states (400 and 380 nm), which exhibit photochromism22. Detailed functional characterization of KnChR with spectroscopy and a structural study together with mutation analysis would be needed to reveal the mechanism of the functional modulation by the cytoplasmic domain.In this study, we report on the electrophysiological characterization of a newly identified multi-domain cation channelrhodopsin from ned Fig. . In othened Fig. . Furtherned Fig. . Therefoned Fig. . Althougλmax) at 430 and 460 nm depending on the protein length  that displayed an action spectrum of λmax = 445 nm, and an absorption maximum of 437 nm after purification (in solution)23. They ascribed the blue-shift to amino acid residues G142 and A146 in 4-TM (T159 and G163 in CrChR2). The replacement of these residues resulted in a blue shift when tested with C1C2 and iC+ +25. KnChR conserves A146 (as A161) while G142 is replaced by A157. Thus, A161 would be one of the color determinants in KnChR.The action spectrum of KnChR, assessed from a voltage clamp experiment, revealed its absorption maxima    (PCa2+/PNa+ = 0.24)27. As PsChR D139H exhibited enhanced Ca2+ conductance, it has a great potential for engineering even higher Ca2+ permeable KnChR26.The comparison of permeability ratio indicates that KnChR is highly Na15. It would be interesting to delineate the functional role of these predicted phosphorylation patterns of KnChR in a native system.As for the physiological role of the cytoplasmic domain, phosphorylation site prediction of KnChR suggested that several motifs may be involved in phosphorylation  of algal groups but not in chlorophyte group28. This algal peptidoglycan layer is similar to peptidoglycan present in bacterial cell wall since it was sensitive to antibiotic (penicillin) and D-cycloserine (inhibitor of D-Ala: D-Ala ligase). The peptidoglycan layer serves as an essential system for chloroplast division in K. nitens29. Blocking of peptidoglycan generation arrested cell division and inhibited chloroplast division. Also, the presence of peptidoglycan biosynthetic pathway genes such as FtsZ3 (Filamentous temperature-sensitive Z) involved in chloroplast maintenance and division in Streptophyte strengthens the presence of FimV domain in this system (K. nitens)31. Thus, the presence of FimV domain with a light sensor is unique and its role via light input is an open question. Interaction of the FimV of KnChR with peptidoglycan and its effect needs further experimental evidence.Moreover, the presence of a FimV domain of KnChR at the cytoplasmic side implies that it might interact with the peptidoglycan layer. Previous studies suggested the presence of peptidoglycan layer around the chloroplast in glaucophyte and streptophyte  were subcloned into a phKR2-3.0-eYFP plasmid ) using an In-Fusion HD cloning kit . Length variants of KnChR  were created by using appropriate PCR primers. For the immunostaining experiment, N-QKLISEEDL-C  in the C-terminal of eYFP was inserted in the plasmid pKnChR (697 amino acids)-3.0-eYFP using inverse PCR. Site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit . All the constructs were verified by DNA sequencing . All the PCR primers used in this study were summarized in Supplementary Tables trans retinal and 5% fetal bovine serum, and under a 5% CO2 atmosphere at 37 °C. The expression plasmids were transiently transfected by using the FuGENE® HD Transfection Reagent  according to the manufacturer’s instructions. Electrophysiological recordings were then conducted 24–36 h after transfection. Successfully transfected cells were identified by eYFP fluorescence under a microscope prior to measurements.Electrophysiological assays and immunostaining of KnChR and CrChR2 were performed on ND7/23 cells, which are hybrid cell lines derived from neonatal rat dorsal root ganglia neurons fused with mouse neuroblastoma. ND7/23 cells were grown on a coverslip in Dulbecco’s modified Eagle’s medium  supplemented with 2.0 μM of all-ND7/23 cells were cultured on glass coverslips. Cells expressing KnChR or KnChR bearing the c-Myc epitope tag at the C-terminus were fixed in 4% paraformaldehyde phosphate buffer solution for 15 min at room temperature. Cells were then washed three times with phosphate-buffered saline (PBS). When necessary, cells were permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Cells were treated with a blocking buffer consisting of 3% goat serum for 60 min at room temperature. After blocking, the cells were incubated with rabbit anti–c-Myc primary antibody  at a 1:500 dilution for 60 min at room temperature, then washed three times with PBS and labeled with goat anti-rabbit IgG secondary antibody Alexa Fluor 594  at a 1:200 dilution for 2 h at room temperature. After a final wash with PBS, the coverslips were mounted on glass slides with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific).2 atmosphere at 37 °C. The cells were washed two times with PBS before fixing with 4% paraformaldehyde phosphate buffer solution for 15 min at room temperature. The cells were washed three times with PBS before labeling with goat anti-rabbit IgG secondary antibody Alexa Fluor 594 at a 1:200 dilution for 2 h at room temperature. After a final wash with PBS, the coverslips were mounted on glass slides with ProLong Diamond Antifade Mountant.Live cultured KnChR bearing the c-Myc epitope tag at the C-terminus expressing-cells were washed with PBS. The cells were moved from the culture medium to serum-free DMEM and incubated with rabbit anti–c-Myc primary antibody at a 1:500 dilution for 60 min under a 5% CO32.Fluorescent images were acquired with fluorescent microscopy LSM880 equipped with ×40 objective lens  and a software ZEN (Zeiss). The captured images were analyzed with Fiji software7. Data were filtered at 5 kHz and sampled at 20 kHz  and stored in a computer . Pipette resistance was 3–6 MΩ. The standard internal pipette solution for the whole-cell voltage-clamp contained (in mM) 126 NaAsp or NaCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 25 HEPES, 12.2 NMG, and adjusted to pH 7.4 by citric acid. The standard extracellular solution for the whole-cell voltage-clamp contained (in mM) 150 NaCl, 1.8 CaCl2, 1 MgCl2, and 10 HEPES, 10 NMG, 5 glucose, and adjusted to pH 7.4 by NMG. Internal and external solutions for ion selectivity are shown in Supplementary Table 50) was determined by using Igor pro 4.06 software . The permeability ratio for each cation was estimated by the Goldmann–Hodgkin–Katz equation33.All experiments were carried out at room temperature (22 ± 2 °C). Photocurrents were recorded with an Axopatch 200B amplifier  under a whole-cell patch-clamp configurationFor whole-cell voltage-clamp, irradiation at 480 nm was carried out using collimated LED  controlled by computer software . Light power was measured directly by an objective lens of a microscope by a power meter . All action spectra were measured at the same light intensity in the range of 410–650 nm by a xenon light source OSG .N = number of biological replicates. The box-and-whisker plots represent the median (center line), the mean (x), interquartile range (box limits) and 1.5 × interquartile range (whiskers). Data were evaluated with the Mann–Whitney U test for statistical significance unless noted otherwise. Means were judged as statistically insignificant when P > 0.05.Data were presented as the mean ± SEM of all experiments with Further information on research design is available in the Peer Review FileSupplementary informationReporting Summary


O-glycans and to contribute to non-covalent molecular scaffolds facilitating the remodeling of polysaccharidic networks during rapid cell expansion. In this work, the characteristics of the PAC domain are described in detail, including six conserved Cys residues, their spacing, and the predicted secondary structures. Modeling has been performed based on the crystal structure of a Plantago lanceolata PAC domain. The presence of β-sheets is assumed to ensure the correct folding of the PAC domain as a β-barrel with loop regions. We show that PDPs are present in early divergent organisms from the green lineage and in all land plants. PAC domains are associated with other types of domains: Histidine-rich, extensin, Proline-rich, or yet uncharacterized. The earliest divergent organisms having PDPs are Bryophytes. Like the complexity of the cell walls, the number and complexity of PDPs steadily increase during the evolution of the green lineage. The association of PAC domains with other domains suggests a neo-functionalization and different types of interactions with cell wall polymersPlant cell wall proteins play major roles during plant development and in response to environmental cues. A bioinformatic search for functional domains has allowed identifying the PAC domain  in several proteins (PDPs) identified in cell wall proteomes. This domain is assumed to interact with pectic polysaccharides and  Plant cell walls are composite structures mainly made of polysaccharides and proteins. Cellulose microfibrils and hemicelluloses form intricate networks, which are embedded in a pectin matrix . AlthougWallProtDB, www.polebio.lrsv.ups-tlse.fr/WallProtDB/, query with “Ole e1 allergen domain” as a keyword). The name of the PDP family was initially proposed by Baldwin et al. [http://pfam.xfam.org/). The firstly described member of this family was a protein from Nicotiana alata named AGPNa3 [Daucus carota DcAGP1 [Arabidopsis thaliana AtAGP30 [Capsicum annuum CaPRP1 [Gossypium hirsutum GhAGP31 [Petunia hybrida PhPRP1 [O-glycosylated Proline/Hydroxyproline-rich domain, or an extensin domain [Among the newly described CWPs families, the importance of the PAC  domain containing-protein (PDP) family could be stressed because of their presence in many cell wall proteomes  has a trichoblast-specific expression and plays roles in root hair elongation, as shown by the reduction in length of root hairs in the prpl1 mutant [FOCL1  produce stomata without a cuticular ledge, and thus, focl1 mutants display drought tolerance [AtAGP30 (At2g33790) is involved in root regeneration in vitro and in the timing of seed germination [AtAGP30 is expressed in root atrichoblasts under the control of ABA signaling [AtAGP31 is expressed in vascular tissues and repressed by methyl jasmonate at the transcriptional level [O-glycans of its Proline/Hydroxyproline rich domain [Functional studies on several of the 1 mutant . Plants olerance . AtAGP30mination . AtAGP30ignaling . AtAGP31al level . AtAGP31al level . In vitrh domain . These sh domain .Plantago lanceolata containing an N-terminal PAC domain  has been determined, highlighting the importance of β-sheets in its secondary structure [Recently, the crystal structure of the PAC domain of an allergenic protein from tructure . In partIn this article, we aim at giving an evolutive overview of the PDPs throughout the green lineage, from Bryophytes to late divergent plants, such as monocots and dicots. We first define more precisely the PAC domain characteristics in order to retrieve PAC domain sequences from available genomic or RNA-seq databases using a tailor-made bioinformatic script. Since the conservation of the primary amino acid sequences of PAC domains was rather low, and since the presence of β -sheets seemed to be essential for domain folding, bona fide PAC domains were selected according to their secondary structure conservation, and protein alignment was done using a software taking into account secondary structures. Modeling of tertiary structures was done based on the available crystal structure of the Pla l 1 PAC domain. Finally, we could draw a phylogenic tree and sort the PAC domains according to their association with other domains. We could also investigate the occurrence of PAC domains in ancestor organisms.A. thaliana genome using that of the AtAGP31 PAC domain. Altogether, 14 candidate sequences were identified and manually checked for the presence of the six conserved Cys residues: At1g29140, At1g78040, At3g09925, At4g08685, At4g18596, At5g45880, At5g54855, AtAGP31, At5g05500 (PRPL1), At5g15790, At2g34790 (AtAGP30), At2g34700, At4g18596, and At2g16630 (FOCL1). These sequences were then used to identify additional PDPs by sequence similarity in eight other angiosperm genomes: Amborella trichopoda, Brachypodium distachyon, Oryza sativa, Sorghum bicolor, Populus trichocarpa, Eucalyptus grandis, Linum usitatissimum, and Gossypium raimondii. About 50 putative PDPs were collected and manually checked for the presence of the six conserved Cys residues. From this first data mining step, it appeared that the level of conservation of the amino acid sequences of the PAC domains could be low. In particular, except between the two first conserved Cys residues (Cys 1 and Cys 2), the spacing between Cys residues could be variable. Thus, the usual homology-based mining was not sufficient, and an alternative strategy was necessary to obtain exhaustive results for each plant. The alignment of angiosperms PAC domains has allowed calculating the range of spacing between the conserved Cys residues. Then, a tailor-made script based on several points detailed in The overall strategy used for this study is summarized in Bryophyta, Marchantiophyta and Anthocerotophyta) to late divergent plants. Altogether, about 450 putative PAC domain sequences were collected  or JmjC and JmjN domains of transcription factors   . In most4 motifs typical of extensins at their C-terminus were found in Anthocerophyta and Lycopodiales, the first bona fide type 4-PDP was found in Psilotales. There is no such PDP in A. thaliana.The PDPs have been classified according to the domains associated with the PAC domain. Four types were distinguished . Type 1 Amborella trichopoda, which is considered as an ancestor common to angiosperms [Brachypodium distachyon, Sorghum bicolor, Zea mays, and Oryza sativa, as well as in Linum usitatissimum, Populus trichocarpa, and Gossypium raimondii. In Poales like B. distachyon and O. sativa, the genes encoding PDPs could be found in tandem , Cryptophyta (Chroomonas sp), Chlorophyta , Streptophyta   . In manyChlorophyta: 10 proteins were found in C. rheinardtii and one in V. carteri ,which both belong to Chlamydomonales. The Glycine residue located upstream the first Cys residue was always missing, and the PAC domains were associated with Proline-rich motifs of two types: either Serine-(Proline)n or (Proline)n and up to three of them could be found in a given protein. However, the secondary structures of these domains were predicted to be α-helices. In C. rheinardtii, the GP1 and GP2 proteins, which both have Serine-(Proline)n motifs, were described as proteins rich in Hydroxyproline resides forming the insoluble glycoprotein framework of the cell wall [C. orbicularis, we could find another interesting PAC domain candidate, which was associated to Proline-rich motifs but contained seven Cys residues. The highest level of identity/similarity was found with two PAC domains of Musa acuminata: GSMUA_Achr4T17330 (45%/51%) and GSMUA_Achr7T01790.1 (39%/50%). The highest level of identity/similarity with a Marchantiophyta PAC domain was found with the Conocephalum conicum PAC domain ILBQ_2004952 (30%/46%) and the M. polymorpha Mapoly0014s0128 PAC domain (33%/45%). Altogether, the sequence showing the highest level of identity to bona fide PAC domains was found in C. orbicularis. This is consistent with the assumption that the Coleochaetales could be one of the ancestors of the green lineage [We could only find PAC domain-related sequences in ell wall ,28. Furt lineage .P. lanceolata PAC domain [Three-dimensional-models were calculated for 41 bona fide and 9 putative PAC domains, based on the crystal structure of the C domain . The seqP. lanceolata PAC domain [Anthocerophyta (Anthoceros formosa), chosen as an ancestral plant, A. trichopoda as the common ancestor to flowering plants, and two higher plants, Oropetium thomaeum and A. thaliana. All four 3-D models show the expected parallel β-sheets forming a β-barrel and the three disulfide bridges. They also contain loop regions as the P. lanceolata PAC domains. The 3D-structure of bona fide PAC domains seems to have been conserved through the evolution of the green lineage. However, the C. orbicularis protein, which was assumed to be an ancestor of the PDPs in the green lineage, only had three β-sheets, but the three disulfide bridges were at the predefined positions  mannose-binding lectin family [O-glycans in vitro [The PAC domains that have been considered apart because of the prediction of α-helices showed completely different 3D-structures . They exn family . In thisn family . This roin vitro .P. lanceolata PAC domain, and for each of the eight A. trichopoda PAC domains, which were considered as representative of the eight phylogenic clades (see below). Each Cys residue has been replaced by a Ser residue, and the change in stability was determined by MAESTRO  from A. trichopoda to Brassicales.Based on all the criteria described above, 300 PAC domains have been selected for the building of phylogenetic trees . They haA. trichopoda PAC domain: clade A ; clade B ; clade C ; clade D ; clade E ; clade J ; and clade K (Equisetum sp PAC domains). The distribution of the PAC domain sequences of the other species was not clear. PAC domains of Bryophytes were represented in clades A, D, and E, whereas a Tmesipteris parva  PAC domain was found in clade B, and a Phylloglossum drummondii  PAC domain in clades C, and J. Of course, one cannot exclude that PAC domains of plants, which have divergent earlier than Amborellales are still missing since only a limited number of fully sequenced genomes are available. Despite the presence of the key Cys residues and of conserved 3D-structure, the large evolutive distance existing between Bryophytes and A. trichopoda together with a relaxed selective pressure could explain the low sequence identity observed between sequences of Tree I. Indeed, whereas terrestrialization is assumed to have occurred 450 MYA [O-glycans, the PAC domain sequence variability could be correlated with the variability of the cell wall composition from Bryophytes to angiosperms [Regarding Tree I, it is difficult to define clades grouping all the PAC domain sequences because most of the bootstrap values were low . We only 450 MYA , the age 450 MYA . Based oiosperms .A. trichopoda PAC domain was found in each of them. Four clades were specific to higher plants, each of them, respectively, comprised the following A. trichopoda PAC domains: AmTr.v1.0.047.45 (clade F); AmTr.v1.0.068.122 (clade G); AmTr.v1.0.153.4 (clade H); and AmTr.v1.0.019.72 (clade I). Monocot and dicots were represented in all the clades, but clade J comprised a high number of grass PAC domains originating from gene duplication (see above). Interestingly, although the tree has been built up with PAC domains only, they grouped according to their association to other domains: type 1-PAC domains were found in clades A, C, F, H, and I; type 2-PAC domains were grouped in clades D, E, and J, with type 2 W-W domains in clade D; type 3-PAC domains were found in clade G with the exception of three of them in clade H with short Proline-rich motifs at their N-terminus; and type 4-PAC domains were only found in clade D. Thus, it seems that there is a link between the amino acid composition of PAC domains, their secondary structure, and the associated domains. Finally, it seems that all the PAC domains of higher plants have a counterpart in A. trichopoda, meaning that the modern multi-domain structures of the PDPs found in the ten angiosperm clades preceded the emergence of angiosperms.In Tree II, the PAC domains were distributed into 10 clades with high confidence bootstrap values (from 72 to 100) with the exception of clade H (28) , S9. An A search for conserved amino acid motifs was done for the PAC domains of each clade of Tree II. The most significant results were found for clades A, B, D, E, G, H, and I . In eachThe combination of sequence conservation with the accessibility of conserved residues on the protein surface shall hint to functional important sites while conserved residues located in the protein core are more likely important for maintaining the fold. Also, conserved residues in the loop regions may have a functional role, although they are less accessible in the static 3D-structural model as loops are often flexible and may move considerably. We, therefore, defined a representative 3D-model for each clade and obtained the solvent accessibility and secondary structure for each residue and aligned this information with the sequence profiles . Indeed,O-glycans assumed from in vitro studies have been conserved and that the distribution of PDPs in the different plant families reflects differences in cell wall polysaccharides. Regarding the W-W C-terminal domain of the clade D PAC domains, its role remains to be unraveled. It is encoded by a distinct exon and could originate from exon shuffling [The conservation of motifs in PAC domains suggests common biological activities. It is possible to infer that their interactions with cell wall polysaccharides or huffling .http://orchidstra2.abrc.sinica.edu.tw/orchidstra2/orchid_blast.php), genome annotation Databases ([http://genome.microbedb.jp/blast/blast_search/klebsormidium/genes), Phytozome , OneKP ([https://db.cngb.org/onekp/) (see https://www.ebi.ac.uk/Tools/st/emboss_transeq/).The sequences used in this study have been retrieved from different databases, such as Orchidstra 2.0  see . When nehttps://blast.ncbi.nlm.nih.gov/Blast.cgi) program has been used for sequence comparison. Similarities between PAC domain sequences have been calculated using either Blast2seq (https://blast.ncbi.nlm.nih.gov/Blast.cgi) or needle (http://www.bioinformatics.nl/cgi-bin/emboss/needle). The sub-cellular localization of proteins has been predicted with TargetP-2.0 ([http://www.cbs.dtu.dk/services/TargetP/) and the presence of β-sheets and/or α-helices using SABLE ([http://sable.cchmc.org/) and NetSurfP ([http://www.cbs.dtu.dk/services/NetSurfP/). The selected PAC domains starting at the Gly amino acid located three amino acids upstream of Cys 1 and ending at Cys 6 have been aligned using PROMALS3D  to take into account the prediction of α-sheets. The phylogeny has been calculated using MEGA7 ([https://www.megasoftware.net/) with the maximum likelihood option and 500 bootstraps. The presence of the PROSITE  and Pfam  domains have been checked in the retrieved sequences. Inside clades, conserved motifs have been identified using MEME ([http://meme-suite.org/tools/meme) or WebLogo3 ([http://weblogo.threeplusone.com/).The BLAST  folds, from which the overall fold is built. The final model is not determined by a single template. As such, it should be better applicable for PAC domain sequences with low similarity to the Pla I 1 PAC domain.C. orbicularis PAC domain appeared as a possible candidate. The association of the PAC domain with Pro-rich sequences seemed to be an ancient event, the most ancient sequence carrying both a PAC domain and a Proline-rich domain being found in Bryophytes, and those carrying both a PAC domain and extensin domains in Psilotales. Despite a great amino acid variability between PAC domains, the tertiary β-barrel structure strengthened by three disulfide bridges has been conserved in all bona fide PAC domains. Finally, the subset of PAC domains belonging to Clade C is intriguing. Their very high level of conservation at the amino acid sequence level suggests that they play critical roles in plant cell walls. Defining the specificity of interaction of the different PAC domains with other cell wall polymers will be one of the next challenges to fully unravel the roles of PDPs in the cell wall architecture.This study has allowed better defining PDPs by combining amino acid sequences features, secondary structures, and 3D-modeling. This protein family has appeared early during the evolution of the green lineage. It has, however, not been possible to identify with certainty a PAC domain ancestor in the presumed precursor organisms of the green lineage even if the


Piglet pre-weaning mortality (PWM) is one of the biggest problems regarding sow performance and piglet welfare. Recently, PWM has increased in some countries, but it is not known if there are similar increases in other countries, nor whether increased PWM is related to either increased numbers of piglets born alive (PBA) or to sow herd size. So, the objectives of the present study were 1) to explore the trend in PWM in Spanish sow herds over a recent 10-year period, along with related measurements such as PBA, stillborn piglets, herd productivity and herd size; and 2) to examine the relationships between PWM and the related measurements.Herd-level annual data from 2007 to 2016 for 91 herds in Spain were abstracted from a sow database compiled by a veterinary consultancy firm that asked client producers to mail data files on a regular basis. The database software automatically calculated herd-level PWM (%) as follows: the total number of piglets born alive to a sow completely weaned during a year (TPBA) minus the total number of piglets weaned by the completely weaned sow during the year divided by TPBA × 100. All the statistical analyses were performed by using SAS University Edition. A growth curve model was applied to incorporate correlations for all of the observations arising from the same farm.P <  0.05). Higher PWM was associated with more PBA, more stillborn piglets and small-to-mid herds . Also, there was a significant interaction between the herd size groups and PBA for PWM (P <  0.05): as PBA increased from 9 to 14 pigs, PWM increased by 9.6% in small-to-mid herds, compared with an increase of only 6.6% in large herds (> 570 sows). Furthermore, as PWM decreased from 18 to 8%, herd productivity measured as PSY increased by 2.2 pigs in large herds, compared with only 0.6 pigs in small-to-mid herds.Over the 10 years, herd means of PWM (standard deviation) increased from 11.9 (4.1) % to 14.4 (3.2) %, and mean PBA increased by 1.9 pigs. Mean age of piglet death during lactation increased by 3.8 days, and later years were significantly associated with herd size and the number of piglets weaned per sow per year (PSY; Large herds were better than small-to-mid herds at alleviating the association between increased PBA and increased PWM. Also, the relationship between decreased PWM and increased herd productivity was improved more in large herds than in small-to-mid herds. Piglet pre-weaning mortality (PWM) is one of the key components in the productivity tree of piglets weaned per sow per year (PSY) when PSY is used as an integrated measurement of reproductive performance in sow herds –3. Over Furthermore, it has been widely reported that most piglet deaths occur at 0–1 days after birth , 16. HowA veterinary consultancy firm  has requested all client producers to mail their data files on a regular basis and has accumulated a sow database. In the summer of 2017, 91 Spanish herds that had 10-year records from 2007 to 2016 were chosen from the database, and their retrospective herd-level annual data for these years were abstracted from the data files.Between 2007 and 2016, most of the studied herds used farrowing crates in farrowing barns. Also, the lactation and gestation diets in the herds were formulated using cereals  and soybean meal. Replacement gilts in the herds were either purchased from breeding companies or were home-produced through internal multiplication programs.Annual PWM values (%) from 2007 to 2016 were calculated by the recording software  using the following equation: the total number of piglets born alive to a sow completely weaned during a year (TPBA) minus the total number of piglets weaned by the completely weaned sow during the year divided by TPBA × 100. Also, the herds were categorized into two groups  based on the 50th percentile of the mean herd size over the 10 years (570 sows). The 50th percentile was chosen as the cut-off point to objectively categorize the studied herds into two groups.Additionally, herd-years was defined as the number of herds times the number of years containing each herd’s annual data. The studied herds were initially chosen because they were thought to have full 10-year records, so the expected herd-years was 910 (91 herds × 10 years). However, some herds had missing or unreliable data in some years, so these herd-years were excluded (see next section).There is a place in the software where producers have the option to record the date that a piglet died and the number of piglet deaths. However, sometimes producers did not record this data or did not record it correctly. As a result, 97 of the 910 herd-year records were excluded: 49 (5.4%) herd-year records were excluded because they had no recorded age for piglet death, and another 48 herd-year records (5.3%) were excluded because the recorded mean age of piglet death was the same as the mean weaning age, suggesting inaccurate recording of the data.There are two types of PWM data: a herd-level value calculated by the software, and an individual value for each sow derived from the piglet deaths for each sow recorded in the software by the producer. The herd-level PWM calculated by the software was used for all PWM analyses. However, for the analysis on age of piglet deaths, we used the death events recorded by the producers because there was no herd-level software calculation for the age of piglet death.P <  0.01).The two PWM values were compared for herd-level internal consistency. The mean PWM calculated by the software was 12.8 (3.6 standard deviation: SD) % ranging between 1.9 and 24.8%, whereas the mean PWM recorded by the producers was 11.2 (3.9 SD) % ranging between 1.2 and 22.4%. In the dataset, the proportion of individual sow PWM values recorded by the producers was 87.5% of the PWM values calculated by the software. Pearson correlation coefficients between the two types of PWM for each of the 10 years ranged between 0.60 and 0.77 . Pearson correlation analysis between PWM and other measurements was performed for each of the 10 years using the CORR procedure. Also, a Chi-square test was performed to examine whether or not the frequency distributions of age of piglet death differed between years. The associations were considered significant when the A growth curve model with random intercept and slope was applied using the MIXED procedure to incorporate correlations for all of the observations arising from the same farm , 19, usiWhen sow herd size and age of piglet death were examined as outcome variables, the data were first converted using a square root transformation because those data were not normally distributed. Model 2 used repeated measures analysis to compare PWM and other performance parameters between the two herd size groups. The model included herd size groups and year as fixed effects.P > 0.10).Model 3 examined the associations between PWM or age of piglet death and possible explanatory factors. The model included the following fixed effects, PBA, squared PBA, herd size groups, year, squared year, the number of stillborn piglets and six two-way interactions between the four main factors . Two possible factors (i.e. weaning age and gestation length) were not included in Model 2 because the correlations between these two factors and PWM were not consistent over the 10 years . The adequacy of the model assumptions for the random effects was checked by visual inspection of normal-probability plots .Mean PWM increased from 11.9 (4.1 SD) % in 2007 to 13.1 (3.2 SD) % in 2016, and PBA increased from 11.2 (0.8 SD) to 13.1 (1.1 SD) piglets over the same period (Table P <  0.05). Additionally, the linear expression of year was found significant for age of piglet death (P <  0.05). However, the cubed year expression was not significant for any measurement (P > 0.10).Table P <  0.05; Table P <  0.05). Figure More PBA, more stillborn piglets and small-mid herds were associated with higher PWM .There were some differences between the two herd size groups, with large herds having lower PWM, more piglets weaned, higher farrowing rates and more PSY than the small-to-mid herds  but not for piglets weaned (P = 0.14). Figure Lower PWM was associated with more piglets weaned and more PSY . Over the 10 years, the age of piglet death in small-to-mid herds increased by 3.3 days, whereas in large herds it increased by only 2.7 days  to 5.6 5.1 SD) days over the 10 years . This difference in the associations between PWM and PSY and between PWM and piglets weaned can probably be explained by the fact that sows in large herds have better fertility performance, such as higher farrowing rates and more litters farrowed per sow per year.. Therefore, in addition to decreasing PWM, it is critical for small-to-mid herds to improve the fertilty performance of their sows and increase their herd productivity, including measures such as PSY.With regard to the limitations of this study, the relationships found between PWM and other variables should be interpreted as associations, not as evidence of causality because our study is a retrospective observational study and not a controlled experiment. Also, the results may not be applicable to all Spanish herds because the studied herds were not randomly selected across from the whole population.Additionally, our study used the herd as the observation unit to explore the relationship between PWM and other reproductive performance measures over 10 years. We believe that herd-level analysis is appropriate because of herd-level changes in management, health conditions and genetics that might have occurred over such a long period. However, it was not possible to perform multivariate analyses to account for any variation due to parity, season, other possible factors or the herd-entry year of sows, and any interactions with these factors. Also, our results might show a herd-level association between variables which might be different from the association that exists at the sow-level . HoweverAdditional file 1. Correlation coefficients of relationships between pre-weaning mortality and performance-related measurements.Additional file 2. A comparison between two herd groups for pre-weaning mortality and other reproductive performance measurements in 91 herds.