Neisseria meningitidis, an obligate pathogenic bacterium in humans, has acquired different defense mechanisms to detect and fight the oxidative stress generated by the host’s defense during infection. A notable example of such a mechanism is the PilB reducing system, which repairs oxidatively-damaged methionine residues. This review will focus on the catalytic mechanism of the two methionine sulfoxide reductase (MSR) domains of PilB, which represent model enzymes for catalysis of the reduction of a sulfoxide function by thiols through sulfenic acid chemistry. The mechanism of recycling of these MSR domains by various “Trx-like” disulfide oxidoreductases will also be discussed. Neisseria meningitidis is the infectious agent responsible for meningitis and septicemia. Its pathogenicity depends on its ability to resist activated oxygen and nitrate species produced by host macrophages in response to infection. For this, N. meningitidis has developed multiple antioxidant defense systems, including the PilB reducing system, which is capable of repairing the sulfoxide form of oxidatively-damaged methionine residues.pilB gene was initially identified in Neisseria gonorrhoeae in 1988, and its name derives from early studies that suggested that PilB was a transcriptional regulator inhibiting pilin synthesis .,18.17,18ructures ,20,21, tIn terms of catalysis, the mechanism involves the deprotonation of the catalytic Cys and protonation of the oxygen atom of the sulfoxide function of the substrate, in order to facilitate the nucleophilic attack of the thiolate and the formation of a sulfurane-type transition state . These pAB use Trx, whereas this role is played by the N-ter in the periplasm  a hydrophilic pocket involving the stabilization of the protonated sulfoxide function of the substrate via hydrogen bonding; and (2) a hydrophobic pocket consisting essentially of the side chain of a Trp residue, which stabilizes the ε-methyl group of the substrate via its indole ring  of PilBAB under steady-state conditions was in fact found to be similar to that of the discrete enzymes in the presence of their preferred substrates. Thus, the fusion does not significantly alter the catalytic efficiency of the MSRs, suggesting that the two function as independent domains even within the context of PilB [S. oneidensis and T. denticola [The catalytic efficiency . In N-ter domain in the periplasm. The N-ter domain of PilB is a disulfide oxidoreductase of the “Trx-like” family, with a TlpA fold and a WCPLC disulfide center redox potential of −0.230 V. Isolated N-ter domain was shown to reduce both isolated MSRA and MSRB domains [k2 approximately 104 M−1.s−1). In this later case, the dissociation of the reduced MSRB/oxidized N-ter complex remains rate-limiting, as in the case of Trx. For MSRA (k2 approximately 102 M−1.s−1), the formation of the oxidized MSRA/reduced N-ter complex occurs more slowly and is in fact rate-limiting, probably reflecting the existence of a structural fit between the oxidized MSRA domain and the reduced N-ter domain (data not shown). Although the structure of the Nter/MSR complexes are not known, the interactions seem to be limited to the active site area as described for other complexes involving “Trx-like” oxidoreductases.In order for the catalytic activity to be regenerated, MSRs need to be reduced at the end of the catalytic cycle, a function performed by Trx in the cytoplasm and the  domains , althougN-ter domain revealed the presence of a rigid 99FLHE102 loop covering one edge of the active site, in both its oxidized and reduced states [k2 values) for the reduction of the MSR domains by a truncated form of the N-ter, in which the loop has been genetically removed, is 10-fold less efficient relative to the intact N-ter [X-ray diffraction and NMR studies of the d states ,33. Thisct N-ter .N-ter domain efficiently reduces only the MSRB domain via an intramolecular mechanism, whereas the reduction of the MSRA domain depends on the classical Trx-like intermolecular mechanism (data not shown)  . The fordis PilB . The preenticola , suggestR-O, but the oxidation of Met leads to an equimolar mixture of R and S isomers, PilB should only be able to efficiently regenerate the half of the Met that is oxidized. Thus, either this activity is sufficient for the bacterium to transiently resist oxidative stress, or the reduction of the S isomer, although quite slow, is sufficient to restore the function of oxidized targets. A final possibility is that reduction of the MSRA domain involves another periplasmic “Trx-like” protein, opening up the possibility to efficiently regenerate both the Met-O stereoisomers.In conclusion, the in vitro results suggest that the mechanisms for reducing the MSRA and MSRB domains in PilB are different: the reduction of MSRB occurs intramolecularly, while MSRA is regenerated intermolecularly. As to the situation in vivo, PilB is a periplasmic protein that deploys its MSR activities to reduce the Met residues of oxidized periplasmic targets. In vivo, the reductive regeneration of the two MSR domains must therefore be efficient in the periplasm to allow the bacterium to cope with oxidative stress. Based on in vitro data, however, the reduction of MSRA is expected to be slower relative to MSRB. As the MSRB domain is specific for the reduction of Met-N-terminal domain, called nDsbD [N. meningitidis nDsbD is able to reduce periplasmic “Trx-like” such as the N-ter domain [k2 values of 6 × 105 M−1 s−1, suggesting the existence of specific recognition between nDsbD and N-ter. The formation of the nDsbD-N-ter complex requires the opening of the nDsbD “cap-loop” region which is positioned above the N-ter redox center. This conformational adaptation would be a prerequisite for the formation of a catalytically-competent complex leading to the formation of an intermolecular disulfide bond between nDsbD and the “Trx-like” partner. It should be noted that N-ter FLHE loop does not seem to be essential for the formation and stabilization of the complex.To efficiently reduce oxidized Met, PilB has to itself be reduced in the periplasm. This role is fulfilled by the disulfide-bond formation (Dsb) D protein, the general transmembrane redox hub protein that uses the reducing power of Trx transferred to periplasmic “Trx-like” disulfide oxidoreductases via specific interactions with its ed nDsbD   [nDsbD efficiently reduces the  partner . Howeverds 4–22) ), the alN. meningitidis was a model study of choice in many respects. The MSR domains of PilB, which have been the subject of extensive investigation at both the kinetic and structural levels, represent model enzymes for catalysis of the reduction of a sulfoxide function by thiols through sulfenic acid chemistry. The PilB protein is able to reduce Met-O in the cytoplasm but also in the periplasm, a compartment particularly subject to oxidation due to the permeability of the outer membrane, by a unique molecular mechanism combining the catalytic advantages of modular proteins, conformational flexibility and a Trx-like recognition mode.During the past decades, the MSR system of N. meningitidis suggested that it might be a good therapeutic target, in particular for the treatment of serogroup B infection for which no vaccine is available. However, the similarities between bacterial and host MSR enzymes could limit the success of this strategy. In this context, bacterial specific periplasmic Trx-like proteins, such as the N-ter of PilB or TlpA2 , may be


This development is generating a great deal of exciting and promising research activity, as evidenced by this Special Issue of Biomolecules. In this paper, we provide both a steady-state and time-dependent mathematical description of a sequence of reactions: superoxide generation, superoxide dismutase, and hydrogen peroxide catalase and ceria regeneration. This sequence describes the reactive oxygen species (ROS); superoxide, O2–, molecular oxygen, O2, hydroxide ion OH– and hydrogen peroxide, H2O2, interacting with the Ce3+, and Ce4+ surface cations of nanoparticle ceria, CeO2. Particular emphasis is placed on the predicted time-dependent role of the Ce3+/Ce4+ ratio within the crystal. The net reaction is succinctly described as: H2O2 + 2O2– + 2H+ → 2H2O + 2O2. The chemical equations and mathematical treatment appears to align well with several critical in vivo observations such as; direct and specific superoxide dismutase (SOD), ROS control, catalytic regeneration, ceria self-regulation and self-limiting behavior. However, in contrast to experimental observations, the model predicts that the 4+ ceric ion state is the key SOD agent. Future work is suggested based on these calculations.The world of medicinal therapies has been historically, and remains to be, dominated by the use of elegant organic molecular structures. Now, a novel medical treatment is emerging based on CeO The applications space is diverse; optical lens polishing, chemical/mechanical planarization of semiconductor wafers, diesel oxidation catalyst, catalytic converters for internal combustion engines, solid oxide fuel cells and sensors , causing the subsequent disease state in the body. Reactions (2) and (3) describe the superoxide dismutation reaction that destroys 3+ to Ce4+. Reaction (4) describes the hydrogen peroxide catalase reaction and regeneration of Ce4+. Note that these reactions are, individually and overall, balanced as they obey both the conservation of mass and conservation of charge. They represent one posssible complete cycle of a disease progression from generation to destruction of the ROS species, O2– and Wherein, reaction (1) represents the generation of /dt = 0,At a steady-state solution, with d [CeLet us consider two limiting cases.2–] >> [H2O2] (the disease state): Here, [Ce3+]/[Ce4+] = k2/k3[H+] and the ceria therapy is dominated by the ratio of dismutation rates . Note, also by rearrangement, we see from this that [Ce3+][H+]k3 = [Ce4+]k2, the ceria redox system is in balance. That is to say, cerous and ceric ions are being created and destroyed in balance (conserved) and the system is self regulating.Case 1. [O2O2] >> [O2–] : Now [Ce3+]/[Ce4+] = k2[O2–]/k4[H+][H2O2 ] and the 4+ state dominates and not much occurs ; it is self-limiting, which is beneficial since it is not needed.Case 2. , since it only appears as a product of the reactions and is present in great excess. Thus, its concentration does not change appreciably with time. The presence of water is manifested through its dissociation constant, which provides both H+ and OH– concentrations, which are critical to the overall set of reactions. Taking these facts into account, we simplify the system into the following set of equations:Further simplifications in the system of equations can be made once we note that none of the equations depend on t = 0. For example, we assumed that We also assumed that no reactants are being introduced or removed after c”. For example, the scale for oxygen is After finding a simplified system, we non-dimensionalize where each variable’s characteristic scale is denoted with a subscript of “In order to reduce the total number of parameters, scales were chosen that use ratios of rate constants and the total ceria concentration to reduce the number of coefficients that simplify the problem. The scales we found are:The resulting system is:From here on, all references to the equations and variables refer to their non-dimensional form. When referring to a variable, we interpret it as the variable relative to its scale. For example, superoxide represents the concentration of superoxide relative to the total concentration of ceria, peroxide represents the concentration of peroxide relative to the ratio of We are interested in both the transient and steady-state behavior of the mathematical model. For the latter, we set d/dt = 0 in the above equations and find admissible solutions for the nonlinear system of equations. In this manner, we seek analytical, long-time solutions of the dynamical system, which provides the possible states to which the transient equations will evolve.2–. The three solutions, with S* nonzero, are: , , and . The remaining five “ideal” solutions are: , , , , and .Let  represent the steady-state solutions to the model. We have found eight possible solutions, three of which correspond to S* not necessarily 0, and five for which S* is identical to 0, which is the ideal scenario since S* corresponds to excess O3+. In the ideal cases, we note that the concentrations of Ce3+ and Ce4+ can either be 1, 0, or anywhere in between, given by the free parameter C*. Thus, it is possible that a steady solution with O2– = 0 could correspond to Ce3+ = Ct, i.e., all ceria is Ce 3+, or all Ce 4+.There are several important things to note. First, C* = 1 in all of the non-ideal cases, which means that the total ceria concentration at steady-state is comprised of Ce3+ and Ce4+ convert between each other. Thus, in order to have a better chance at avoiding the non-ideal cases, and to allow as much flexibility as possible in the selection of steady states where O2– goes to 0, a possible ideal initial dosage would be one completely comprised of Ce4+, i.e., starting as far away as possible from the non-ideal cases.In the chemical reactions, CeWe approximated the system of differential equations with MATLAB’s ode45 integrator that implements the Runge–Kutta method.3+; therefore, the crystal would be completely Ce2O3 or in the case of ceric ion Ce4+, CeO2. What would the results look like in these scenarios?The main goal is to eliminate as much of the excess superoxide in the system as possible. In all results below, we have used 3+ does not reduce the amount of O2– at all. There is a slight increase in the amount of superoxide before it decreases back to its initial amount as the amount of Ce3+ recovers  back to its original level. The Ce3+ is somewhat ineffective in controlling O2–.4+ is present in the crystal, i.e., CeO2. We see that all of the superoxide is eventually eliminated. Although there is a similar small increase in the amount of the superoxide, its concentration diminishes along with 3+, while none of the Ce4+ remains. The system is very active in controlling superoxide, unlike the results in Now, consider 2O2 (not shown), as well as an increase in O2. From these initial plots, it appears that the ceric ion is much more effective than the cerous ion at reducing the superoxide anion concentration.In both 4+. As was calculated in the non-dimensional equations, many of the terms include ratios of rate constants. We now consider what would happen if one of these two rate constants were greater than the other and what would happen if those reactions occurred at different rates.From this knowledge of the importance of the +4 state, in the next few cases, we consider starting with only an initial concentration of Cek1 is the dominating rate constant, that is, the production of superoxide overpowers the rest of the system and a serious disease state exists.In 3+/Ce4+ does not seem to be responding over time and while The plots in 3+/Ce4+ redox couple.+4 when superoxide is initially present.The next few figures look into the dosage amount of ceria that should be used in relation to the amount of excess superoxide that is present. Does introducing a much greater amount of ceria have an overall effect on the rate of control of superoxide? What about the opposite scenario, in which a smaller amount of ceria is initially used? Will the excess superoxide be controlled in a reasonable time scale? In 3+ initially present ) and in reducing hydrogen peroxide ). The prevailing general belief that the 3+ state is critical to addressing super oxide dismutation chemistry, while the 4+ state is hydrogen-peroxide-catalase-dominant may stem from the nature of the ceria crystals being studied. Relatively large (10–15 nm) highly agglomerated ceria crystals will in fact be highly depleted in the 3+ ion concentration as has been shown by Hailstone [The main goal for the model was to show the qualitative role of rmohamed  on 5–10 rmohamed , that seailstone  and, the2– is converted to O2. When 2– never goes to zero in this scenario. These two extreme cases of the relative rate constants of generation and recovery from the disease state reflect on what could be interpreted as a dose–response behavior that has been observed previously [We also determined that the value of eviously ,28.2– excess. This is immediately evident by inspection of the overall equation: H2O2 + 2O2– + 2H+ → 2H2O + 2O2.It was also found that the amount of 2–. This trend could possibly be very cost effective since much smaller amounts of 2– with minimal negative effects although one would not want to get outside the range of normal physiological pH values (6.7–7.2).Introducing ceria with the correct amount of The model was constructed to the qualitative behavior of the chemical reactions. We note that the model does not account for biological processes that modify the chemical composition of the environment in which the model is set. For example, the circulatory system is constantly introducing oxygen to the reaction environment, and mitochondria are constantly converting oxygen into metabolic products. Both of these phenomena become significant above the time frame of seconds, but their precise effects on the chemical model are unknown. These effects, as well as species diffusion, incorporation of other reactive oxygen species, as well as reactive nitrogen species, could be the subject of future model refinements.According to the model, the production of superoxide is limited by the initial concentration of hydroxide, and the destruction of superoxide is limited by the initial concentration of hydrogen ions  goes away , the entire reaction sequence shuts down .The system is catalytic .The system is both self-limiting and self-balancing.It appears to directly control reactive oxygen species and specifically superoxide, perhaps due to the nature of the oxygen vacancies within the crystal.Within the framework of the reaction sequence as described, the dominant ions are the cerium 4+ state and H+ initially but in totality, the chemical reaction system requires both 4+ and 3+ cerium ions, which are constantly in balance and equilibrating.This self-consistent system of equations and the resultant net equation seem to connect quite strongly, qualitatively with observations made in the laboratory. This is both gratifying and encouraging. To wit:


Because overall cranial morphology-biomechanics linkage in carnivorans is significantly influenced by both feeding and non-feeding ecological variables, whole-skull mechanical performance measures may be less sensitive to feeding ecology than regional characteristics within the skull. The temporomandibular joint could be one regional characteristic that is highly sensitive to feeding ecology considering that this joint is used in prey capture, food processing, and experiences compressive loading during mastication. Through 3D model construction, 3D printing, and compression tests, morphological and mechanical performance measures were determined for the temporomandibular joint trabecular bone structure of 40 species representative of the phylogenetic and ecology diversity of Carnivora. Remarkably, the results indicate that relative fill volume, relative structural complexity, elastic modulus, and relative maximum compressive strength of trabecular bone structure are not significantly related to phylogeny or ecology. The results reveal that morphological and mechanical performance attributes of trabecular bone structure are primarily influenced by body size, and that positive centroid size allometry and positive body mass allometry are present for structural complexity. The lack of feeding ecological signal in dorso-ventral compressive loading of temporomandibular joint models indicates that carnivoran temporomandibular joint trabecular structures may not undergo significant differential remodeling as an evolutionary response to different mechanically demanding feeding tasks. Historically it has been assumed that cranial structure-biomechanics relationships are primarily influenced by feeding ecology in carnivorans, but recent results from geometric morphometrics and finite element analysis suggest that carnivoran cranial morphology is related to both feeding and non-feeding variables . The carSus (domestic pigs) shows that the mandibular condyle is subject to loading during mastication and that these forces are mostly compressive . Hig. Hig2]. Trabecular structure 3D models were imported into 3D Systems Sprint printing software. The specimen number was engraved on the superior surface of a given sample with a depth of 0.1mm. Samples were then printed using a 3D Systems ProJet MJP 2500 3D printer. All samples were printed using VisiJet M2R-CL material, which possesses a tensile strength of 35–45 MPa . Althoug3) and relative structural complexity measured as relative surface area (mm2) were calculated using the 3D model that served as the basis for each 3D print. Raw data from compressive load tests were analyzed using Excel to obtain elastic modulus (MPa) and relative maximum compressive strength (N) for the trabecular bone structure of each species using the stress-strain curves of each sample test. Stress was calculated by dividing the recorded force by the surface area of the cylinder face  in contact with the compression plate of the testing machine. Strain was calculated by dividing the recorded vertical displacement distance (mm) by the original height of the cylinder sample (10 mm). Maximum compressive strength was then determined by identifying the maximum stress value. Elastic modulus was calculated by estimating the slope of the linear (elastic) region of the stress-strain curve. Because the test protocol involved standardizing the size of the test samples, centroid size information and body mass information were reintroduced into the test data by multiplying relative fill volume, relative structural complexity, and relative maximum compressive strength each by centroid size [Morphological data describing trabecular bone structure were measured using Geomagic Wrap. Relative fill volume  because surface area increases within 2 dimensions while body size increases in 3 dimensions. It was expected that m = 1 for maximum compressive strength because the capacity for withstanding compressive force should increase uniformly with increase to body size given that the amount of material available to withstand loading would increase. Positive/negative allometry was defined as expected slope values failing to fall within the confidence intervals of the estimated slope coefficient from PGLS regression analysis.Phylogenetic generalized least squares (PGLS) analyses were used to assess potential relationships of morphological and mechanical performance data with activity cycle, terrestriality, habitat breadth, dietary breadth, trophic level, maximum longevity, age of sexual maturity, mean monthly precipitation, temperature, dietary mechanical demand, and suborder. The ecological data were obtained from the PanTHERIA database of mammalian ecological characteristics . AdditioData analysis was carried out using R in the phytools, nlme, ape, Geiger, Morpho, picante, geomorph, PHYLOGR, OUwie, and caper packages –18. The Mellivora capensis (honey badger) displays the greatest relative fill volume for a trabecular bone structural sample, whereas Bassariscus astutus (ringtail) has the least relative fill volume  for mandibular condyle trabecular bone occurs in Hydrurga leptonyx  and the least relative structural complexity (269.4243 mm2) is apparent in Fossa fossana . The results indicate that centroid size included structural complexity is positively correlated with maximum longevity and sexual maturity age, as well as negatively correlated with dietary mechanical demand ; a species that wields durophagous dentition and is known to crush the bones of much larger species [Odobenus rosmarus  trabecular bone morphology appears to be very poor for withstanding compressive loading given that this species exhibits the minimum values for elastic modulus and relative maximum compressive strength. This finding may be related to the diet of O. rosmarus that consists primarily of soft invertebrates and to the great body size of this particular species [Galidictis fasciata (broad-striped mongoose) and Ursus arctos (brown bear), respectively.Interestingly, the species with the maximum and minimum values for the morphological measurements  are not the species that exhibit the maximum and minimum mechanical performance values. The greatest elastic modulus and greatest relative maximum compressive strength are displayed by  species , 20. Fro species , 21. IncPotential relationships of structural mechanical performance data were explored using phylogenetic generalized least squares analyses with activity cycle, terrestriality, habitat breadth, dietary breadth, trophic level, maximum longevity, age of sexual maturity, mean monthly precipitation, temperature, dietary mechanical demand, suborder, relative structural complexity, and relative fill volume. As elastic modulus is by definition size independent, size inclusion and size correction are not required for elastic modulus analyses . The resBody mass included maximum compressive strength is positively correlated with maximum longevity, age of sexual maturity, and relative fill volume. Also, body mass included compressive strength is negatively correlated with mechanical demand. Similar to centroid size corrected maximum compressive strength, the only significant relationships for body mass corrected maximum compressive strength are a positive correlation with relative fill volume and a negative correlation with relative structural complexity. Although maximum compressive strength shows correlations with some ecological variables, the analyses that account for body size via centroid size or body mass show that maximum compressive strength only has significant relationships with relative fill volume and relative structural complexity. Overall, these results indicate that increases to elastic modulus and maximum compressive strength are associated with increases to trabecular bone relative fill volume. Conversely, elastic modulus and maximum compressive strength decrease as the relative structural complexity of trabecular bone increases.The degree to which allometry influences trabecular bone of the mandibular condyle is further evaluated through linear regressions of the natural logarithms (ln) of morphological and mechanical performance characteristics versus ln centroid size and ln body mass . The resThe results pertaining to the relationships of morphological and mechanical performance attributes are essentially consistent with expectations. Elastic modulus and relative maximum compressive strength have positive correlations with relative fill volume, which matches expectations because greater amounts of material through which force can be distributed within a standard set of dimensions should be associated with greater stiffness and strength. The negative correlations between relative structural complexity and elastic modulus, as well as between relative structural complexity and relative maximum compressive strength, are also consistent with expectations for how these trabecular bone structures should respond to compressive loading. When adding compressive loads to rigid structures, a highly complex structure made up of numerous smaller load bearing supports would be expected to fail overall shortly after the weakest support undergoes fracture. In contrast, a second structure made up of exactly the same amount of material, but in the form of a completely solid column, should be capable of withstanding greater compressive loading because the overall structure is not dependent on the structural integrity of a smaller component. All in all, the mechanical performance results for trabecular bone structures are consistent with expectations for how those structures function under compressive loading.0). These findings are consistent with results from a study on three platyrrhine primate taxa that suggest mandibular condyle trabecular bone morphological characteristics do not significantly vary despite dissimilar dietary behavior [Although many different structures were evident in carnivoran trabecular bone , neitherbehavior . Neverthbehavior –5.Ursus americanus tibia cortical bone has a tensile strength of 166 to 198 MPa [These unanticipated results might suggest that ecological variables do not require drastically different temporomandibular joint mechanical capabilities in carnivorans. An alternative explanation is the possibility that selective pressures on trabecular bone structure from feeding ecology may actually be diminished by the mammalian mandibular condyle bone remodeling plasticity within an individual’s lifespan . That is 198 MPa . Thus, a 198 MPa . Future The results indicate that centroid size allometry and body mass allometry are significantly correlated with temporomandibular joint trabecular bone structural properties. The positive allometry for structural complexity, which is a morphological attribute that is associated with both lower elastic modulus and lower maximum compressive strength, seems to suggest that carnivoran species of larger body size possess mandibular condyle trabecular bone morphology that is not structurally optimized for withstanding high compressive loads. This observation could reflect the relatively reduced mechanical demands in larger species  and/or osteological growth mechanisms that maintain comparable trabecular bone subcomponent structures regardless of species body size. Body size and other non-feeding variables have been shown to influence the overall cranial morphology of Carnivora  and the Lastly, the findings of this study are valid in a cross-species comparative context given the focus on broad taxonomic sampling (across 14 families) rather than multiple individuals of the same species . Bone functional adaptation theory indicates that the trabecular arrangement reflects stereotypical loading regimes experienced by the individual . TherefoUpon consideration that the temporomandibular joint is involved in prey capture and food processing, that the mandibular condyle experiences compressive loading during mastication, and that biomechanical feedback appears to influence mandibular condyle biomineralization levels and coordination of oral tissue development, the morphology and mechanical performance of this structure appeared to have great potential to provide engineering-based measures of feeding ecology –5. The rS1 Table(CSV)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Octopus vulgaris and present evidence of their expression not only in the brain but also in male and female reproductive organs. We identified three synapsin isoforms phylogenetically correlated to that of other invertebrates and with a modular structure characteristic of mammalian synapsins with a central, highly conserved C domain, important for the protein functions, and less conserved A, B and E domains. Our molecular modeling analysis further provided a solid background for predicting synapsin functional binding to ATP, actin filaments and secretory vesicles. Interestingly, we found that synapsin expression in ovary and testis increased during sexual maturation in cells with a known secretory role, potentially matching the occurrence of a secretion process. This might indicate that its secretory role has evolved across animals according to cell activity in spite of cell identity. We believe that this study may yield insights into the convergent evolution of ubiquitously expressed proteins between vertebrates and invertebrates.Synapsins are a family of phosphoproteins fundamental to the regulation of neurotransmitter release. They are typically neuron-specific, although recent evidence pointed to their expression in non-neuronal cells where they play a role in exocytosis and vesicle trafficking. In this work, we characterized synapsin transcripts in the invertebrate mollusk  They are mostly in the nervous system, but also expressed in various cell types where they play a role in exocytosis and vesicle trafficking as well as they do in neurons5. In a few studies, synapsins have been also identified at the level of the reproductive system, in particular in human spermatozoa and unfertilized zebrafish eggs6. As synapsins closely associate with synaptic vesicles, their involvement in a wide range of secretory events, including those typical of reproductive organs, can be anticipated.Synapsins are a family of phosphoproteins considered ‘key actors’ in the regulation of neurotransmitter release, for a review see11. The recent development of cephalopods’ genomics has provided important tools for a deep understanding of their evolution and comparative investigations with other animal species12. Since then, several molecular pathways involved in processes, such as morphogenesis, have been found common to vertebrates14.The existence of non-neuronal functions of synapsins has been attested and studied in a limited number of animal species. Among invertebrates, cephalopods currently represent an important model system for comparative research of conserved, as well as divergent, animal features due to the acquired neural complexity and morphological noveltiesOctopus vulgaris, with particular attention to their specific localization at the level of brain areas and reproductive organs. We identified three synapsin isoforms phylogenetically correlated to those of invertebrates and with a highly conserved C domain. We present evidence of synapsin expression, not only in the brain, but also in the ovary and testis and further evaluated their spatiotemporal expression at two stages of maturation of the reproductive organs. We found that synapsin expression increases during sexual maturation in cells with a known secretory role and this potentially matches the occurrence of the secretion process. Molecular modeling analysis further provided a solid background for predicting synapsin phosphorylation-dependent binding to ATP, actin filaments and secretory vesicles. These results suggest a role of synapsins in reproductive organ maturation and its possible involvement in vesicle secretion. This protein feature may be conserved across animals and evolved according to the cell function in spite of cell identity.In this work, we aimed at characterizing the synapsin homologues coded and expressed in the invertebrate Octopus vulgaris genome contains a single synapsin gene that is strictly correlated to that of other invertebrates and particularly to the synapsins of other cephalopods such as Loligo pealei and Octopus bimaculoides  yielded two isoforms generated by alternative splicing of the same gene. These two cDNAs result in a long protein, termed Syn-long, and a short truncated protein termed Syn-short of 521 and 487 amino acids, respectively . Moreover, O. vulgaris Syn 8.2 exhibits some substitutions in domain C that are also present in ApSyn8.2, HpSyn, LpSyn, and mammalian synapsins  octopus synapsin A and E domains  isoforms have a higher similarity with that of human SynIa; (ii) octopus synapsin B domain resembles that of human SynIIIa (43% identity); (iii) octopus synapsin C and D domains have a higher similarity with those of human SynIIa/IIb (respectively 66.9% and 64.7%) and SynIIb (60.2% and 63.6%)  for cAMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase I (CaMKI) and IV (CaMKIV) in the A domain18; sites 4 and 5 (Ser62 and Ser67) for mitogen-activated protein kinase (MAPK) in the B domain20; site 8 (Tyr301) for Src tyrosine kinase in the C domain21  and close-neighbor-interchange tree (NJ) search methods. Figure 22).The reproductive apparatus of animals of both sexes  were first staged, to assess their level of maturation, through gross macroscopic and histologic investigations . In immature .To determine synapsin localization and its expression level during sexual maturation in both sexes, an immunostaining assay was performed at mature and immature stages using the anti-synapsin E domain antibody (G-304) previously validated by immunoblot on squid optic lobe extracture Fig.  and matuure Fig.  ovaries.ure Fig.  and matuure Fig.  testis, O. vulgaris synapsin C domain (residues X to Y)25. The threading algorithm identified the C domains of R. norvegicus synapsin II (PDB code 1i7l) and I (PDB code 1pk8) as the best templates for O. vulgaris synapsin C domain. The two template domains show a very high sequence and structural identity, with a Root Mean Square Deviation (RMSD) between them of 0.8 Å. The O. vulgaris and R. norvegicus C domain sequences have 65% identity, and the alignment used to predict the structure extended over 95% of the residues. The best ranking I-TASSER model of O. vulgaris synapsin C domain is shown in Fig. R. norvegicus synapsin II C domain. The model had C-score 0.70  and TM-score 0.81 (60% above correct topology threshold), highlighting the very good quality of the predicted structure (see Methods for a brief explanation of these parameters). The RMSD between the model and the C domains of R. norvegicus synapsins II and I were 0.47 and 0.7 Å, respectively, values typical of very similar folded conformations. Indeed, as can be seen in Fig. R. norvegicus synapsin II C domain, and the structure displays all the characteristic features of synapsin C domains that have been shown to mediate ATP binding. These include charged residues near the (putative) ATP binding site, the multifunctional (MFL) and the phosphate-binding loop (PBL).We used the I-TASSER online software to generate a structural model of the O. vulgaris synapsin C domain has two glutamate residues at the ATP binding site (Glu347 and Glu360), similarly to synapsin I (where the corresponding residues are Glu373 and Glu386) and opposite to synapsin II and III, where the first glutamate is substituted by a lysine . This inversion of amino acid charge has been shown to be responsible for the different regulation exerted by Ca2+ on ATP-binding to synapsins I, II and III28.However, 5. Interestingly, an increasing number of molecules considered neuron-specific, such as Tsga10, tau protein, and cadherin, have been found in both nervous tissue and reproductive organs31.Synapsins are evolutionarily conserved phosphoproteins primarily expressed in the brain and fundamental to the regulation of neurotransmitter release. However, recent studies reported their presence in non-neuronal cell types where they play a role in exocytosis and vesicle trafficking37. This is possible by the cooperative functions of domains specific for actin filament and membrane phospholipids binding. Several functional domains, named from A to J, can be differently assembled and give rise to alternative transcripts. The majority of these domains are highly conserved across vertebrates and even invertebrates39. Domain C is the core of synapsin function and has a crucial role in many of the conserved functions such as actin and phospholipid interaction, ATP binding and phosphorylation39. Domains A, C, and E characterize the prototypical invertebrate synapsins and share high similarity with vertebrates. Domain B is known to be a conserved linker connecting domains A and C. Its specific functions have not been fully described but the presence of two phosphorylation sites for the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) suggests a regulation of synapsin function by this signaling pathway40. These sites are conserved in octopus synapsin as shown in Fig. 41. ALPS are able to insert into curved membranes and have been identified in several proteins including Syn I, Syn II and Syn III42. This region is not conserved in octopus synapsin thus suggesting that protein-targeting may be controlled through mechanisms different from that found in vertebrates. Indeed, the interaction between domain B and C is considered important for the synapsin correct localization43.Although this might seem strange, one should consider that several processes occurring in cells of the reproductive organs during sexual maturation recall somehow the life of a neuron. Starting from their active division, differentiation, and migration, at the beginning of their development, they end acquiring a remarkable secretory function that is at the basis of neurotransmission. All these processes require a well-coordinated rearrangement of the intracellular and extracellular tissue scaffolding and the mobilization of vesicles across cytoskeletal elements mediated by interacting proteins. In particular, the last function is exploited through synapsin ‘sandwich’ association between the surface of synaptic vesicles and cytoskeletal filaments14.When seen from an evolutionary perspective, the acquisition of domains meets the increase in nervous system complexity. Given the highly developed nervous system of cephalopods, these animals might represent a valuable biological model to study synapsin evolution and function. The recent advances in cephalopod genomics have boosted many fields in cephalopod research; it is now clear that, in spite of their evolutionary divergence, several common principles of morphogenesis and physiological processes exist between vertebrates and invertebratesOctopus vulgaris brain and reproductive organs. We first identified three synapsin isoforms, phylogenetically correlated to that of invertebrates; all contained an E domain, a highly conserved C domain and less conserved A, B and E domains.In this study, we characterized the synapsin isoforms coded and expressed in 22, but no data currently supports the existence of a non-canonical AUG in vertebrates and mammals synapsins. Despite this, our study reveals novel insights into the use of non-AUG translation to expand the functional coding potential of octopus genome.Octopus synapsin sequences, as in other mollusks, are characterized by non-AUG canonical start codon. It is well known that translation can start at non-AUG codons across eukaryotesIn the current work, we present evidence of synapsin expression in brain, ovary, and testis and show its increase during sexual maturation in cells with a known secretory role. This potentially matches the occurrence of a secretion process in reproductive organs, such as the release of vitellogenic granules from follicle cells to the oocyte, and the sperm heads acrosomal reaction. Interestingly, one other large family of membrane receptor protein, the G protein-coupled receptor (GPCR) is known to act at the interphase between hormonal stimulation and reproductive organ response thus controlling sexual maturation.44. Both octopus ovary and testis are enriched in several GPCR-like adhesion receptor families7, thus opening the possibility that a secretory machinery possibly involving GPCRs is activated in these organs during maturation and functions. Expression of GPCR proteins has been assessed also in male reproductive system in a number of organisms including mammals. GPCRs have been proven to be associated to sperm physiology46 and, in particular, to acrosome reaction through a variety of signaling pathway46.It has already been shown in other invertebrates, that during female reproduction, vitellogenin (Vg) gene expression and the uptake of Vg or yolk protein into the oocyte is dependent on the correct expression and function of GPCRs2+, just like Ca2+-dependent neurotransmitter release. This phenomenon occurs also in cephalopods where a calcium-mediated mechanism induces acrosome reaction through the bulging, vesiculation, and dehiscence of the plasma membrane around the acrosome and the nucleus. It is worth notice that this process resembles the acrosome reaction studied in other species48. Vesicles localization around the sperm nucleus observed in these studies is in line with the perinuclear localization of synapsin found in our samples of testis, although further functional investigations are needed to clarify the specific function of synapsins in sperm activity.The acrosome reaction is a stimulus-induced exocytosis process activated by an increase in cytoplasmic Ca2+-dependent functional binding to ATP, actin filaments and secretory vesicles in a phosphorylation-dependent manner. The available crystal structure of synapsin isoforms from different species revealed that the octopus synapsin C domain is structurally homologous to a family of ATP-binding enzymes27. Synapsins are known to bind ATP with high affinity although with a differential regulation by Ca2+. In particular, while Ca2+ activates ATP binding to synapsin I, it does not affect ATP binding to synapsin II, and inhibits ATP binding to synapsin III27. In synapsin I, the Ca2+ requirement for ATP binding is mediated by an evolutionarily conserved glutamate residue (Glu373) at a position where synapsins II and III contain a lysine residue . Similar to synapsin I, octopus synapsin displays a glutamate residue (Glu347) in that position, pointing to a Ca2+-regulated ATP binding.Interestingly, our sequence and molecular modeling analysis provide a solid background for predicting synapsin CaThe presence of synapsin in non-neuronal tissues may be the starting point for explaining the role of synapsin-like molecules in simple organisms lacking a nervous system, such as protozoa, where a molecule similar to synapsin, formed only by the C domain, was discovered. In fact, from a functional viewpoint, the C domain is important for both the binding to ATP molecules and the cytoskeletal components of actin.12.Interestingly, the presence in the octopus genome of other genes coding for molecules involved in cell-cell interaction and signaling to the actin cytoskeleton, such as cadherins, and particularly their expression in reproductive organs has been already assessed49. Within this framework, our study might yield insights into their convergent evolution and maintenance of molecular and functional conserved roles across metazoans.Taken together our results point toward the possibility that, in the octopus, a similar regulatory mechanism is involved in the process of vesicle secretion occurring in phenotypically different cells. Although the tissue-specific role of many ubiquitously expressed protein is still unknown in many animal species, recent evidence points toward the conservation of their function in the different cell typeset al.50. All experimental procedures were previously approved by the local Ethical Committee  of the IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Ospedale Policlinico San Martino, Genova, Italy) and by the Italian Ministry of Health (authorization n. 1111/2016-PR) and all experiments were performed in accordance with relevant guidelines and regulations.Specimens of both sexes were collected from local anglers of the ligurian coast of Italy. All our research conformed to the ethical principles of the three Rs  and of minimizing animal suffering, following the Directive 2010/63/EU  and the guidelines from Fiorito 2. Brain supraesophageal mass (SEM), ovary and testis were collected and frozen for RNA extraction or fixed for in situ hybridization (ISH) and immunofluorescence. Total RNA from these samples was extracted using the RNeasy Microarray Tissue Mini Kit (QIAGEN). Following extraction, RNA was treated with RNAse-free DNAse according to the manufacturer’s recommendations to digest contaminating genomic DNA. For each sample, 1 μg of total RNA was retrotranscribed with Superscript III First Strand Synthesis SuperMix (Invitrogen) following the company’s instructions.Animals were anesthetized in cold seawater supplemented with 1% ethanol and 55 mM MgClOctopus bimaculoides  was used to identify homologs of synapsin. A partial sequence resembling the synapsin sequence from O. bimaculoides was identified (JGI: LOC106879677 and NCBI: XM_014929352.1) and used to construct partially overlapping primers to isolate a clone from O. vulgaris . PCR experiments gave the expected synapsin sequence together with other synapsin isoforms , were deposited in NCBI under the following accession numbers: Syn-long: KY768851; Syn-short: KY768852; Syn8.2: KY768853.A blast search by using the available genome of The Vector NTI Suite  software package was used for sequence analysis. Protein sequences of synapsin from several organisms were aligned using ClustalW in the MEGALIGN program from Lasergene .Accession numbers of sequences used in this study and the multi-alignment used to reconstruct the phylogenetic tree are reported in the Additional File Comparison between synapsin domains was performed by sequence identity matrix using GeneWorks   and neighbor-joining (NJ) methods in MEGA 6.0The rate of variation among sites was modeled with a gamma distribution with shape parameter = 1.1358, in an analysis involving 17 amino acid sequences. For the NJ method, evolutionary distances were computed using the JTT matrix-based method and are expressed as numbers of amino acid substitutions per site. Bootstrap confidence limits were obtained by 1000 replicates in both ML and NJ analysis. Tree files were viewed by using MEGA 6.0.A digoxigenin-labeled antisense riboprobe for synapsin was synthesized from cDNAs cloned in the pGEM®-T Easy Vector (Promega) following the manufacturer’s protocol . The length and quality of the DIG-labeled RNA synapsin probe was controlled on an agarose gel. According to manufacturers, 1 μg of probe was added to 3 volumes of RNA sample loading buffer (Sigma-Aldrich) heated to 65 °C for 10 minutes, and then chilled on ice. The probe was loaded onto a TAE/1% agarose/sybrSafe gel next to 1 kb ladder (ThermoFisher Scientific) and run at 80 V.In situ hybridization (ISH) was carried out on O. vulgaris SEM  and mounted with Mowiol. Parallel sections were stained with hematoxylin and eosin (Sigma-Aldrich).One band at the expected length of ≈810 nt is clearly visible . Offline analysis for immunofluorescence quantification was performed using ImageJ with the Corrected total fluorescence method53. Fluorescence intensity in regions of interest was quantified in single section planes (1 µm).Oocyte and testis samples were fixed overnight in 4% PFA in artificial seawater, embedded in OCT compound, serially sectioned on a cryostat and collected onto Superfrost Ultra Plus (Menzel-Gläser). 20 μm-frozen sections were permeabilized in 1 × phosphate-buffered saline (PBS) + 1% Triton X-100 (PBS-T) twice for 5 min at room temperature (RT) and incubated in blocking solution  for 1 h at RT. Synapsin rabbit polyclonal antibodies (G-304 rabbit E-domain specific) were diluted 1:100 in blocking solution and applied overnight at 4 °C. After three PBS-T washes for 10 min, sections were incubated in Alexa Fluor® 488-conjugated anti-rabbit secondary antibody , Alexa Fluor™ 647 Phalloidin , and DAPI/Hoechst (1:1000) 2 h at RT. Tissues were rinsed several times and mounted in ProLong Gold antifade reagent . Sections were imaged by confocal microscopy . O. vulgaris synapsin C domain (residues 91 to 391) was obtained using the I-TASSER structure prediction web server25. I-TASSER is a hierarchical approach to protein structure prediction that ranked first in recent community-wide experiments (CASP7 to CASP12)54. Given a target sequence, it first identifies suitable structure templates in the PDB database based on sequence similarity , and models ab initio the regions for which no template can be found; then, using stochastic simulations, it assembles the template fragments into full-length conformations and refines them. For O. vulgaris synapsin C domain, I-TASSER identified rat synapsin II C domain as the best template (PDB code 1i7l). The two domain sequences show 65% identity, and the alignment to predict the structure extended over 95% of the residues.A structural model of the folded The two parameters used by I-TASSER to estimate the quality of a predicted model are the C-score and the TM-score. The C-score is calculated based on the significance of the template alignments and the convergence parameters of the structure assembly simulations. The C-score is typically in the range , where the higher the value, the higher the confidence of the model. The TM-score is a measure of the difference between two structures that is more sensitive to global similarities than local differences, whose estimate for a predicted structure with respect to the actual (unknown) fold has been demonstrated to be correlated to the C-score. A predicted TM-score > 0.5 indicates a model of correct topology, while a TM-score < 0.17 corresponds to random similarity.Statistical analysis was performed using SigmaPlot 13.0 . Normality of the datasets was first assessed with the Shapiro-Wilk normality test. Student’s t-test was used to assess the difference between two experimental groups. Three technical replicates were used (n = 3). p values < 0.05 were considered significant .Additional File 1Additional file 2Additional file 3Additional file 4Additional file 5Additional info on probe length of additional file.5a


Paragonimus westermani, the oriental lung fluke infecting over 20 million people worldwide, has not been thoroughly investigated thus far. We analysed genetic markers (COI and ITS2) of P. westermani from coprolite specimens (n = 6) obtained from 15th to 18th century Korean mummies. Our results indicated that all P. westermani sequences were generally distinct from the other species of the genus Paragonimus. The sequences were clustered into three groups: Group I for East Asia; Group II for South and Southeast Asia; and Group III for India and Sri Lanka. In this study, we found that ancient P. westermani sequences in Korea belong to Group I, adding invaluable information to the existing knowledge of Paragonimus paleogenetics.The genetic information of ancient  Paragonimus eggs hatch before entering snails (the first intermediate hosts). When the cercariae penetrate crustaceans such as crayfishes or crabs (the second intermediate hosts), they encyst in gills, muscles, and viscera, developing into metacercariae. After definitive hosts (e.g. humans) eat the freshwater crustaceans raw, the metacercariae excyst in the small intestine, penetrating the intestinal wall. They then traverse the diaphragm, entering the peribronchiolar tissues of the lung where they mature into adults within 8-12 weeks.Paragonimiasis occurs in definitive hosts after ingestion of infected intermediate hosts.Paragonimus constitutes a species-rich group from the tropical regions in South and Southeast Asia, Africa, and Central and South America as well as the temperate zone of North America and East Asia.Paragonimus have been reported so far;,,,,Paragonimus, the most common species is P. westermani, the oriental lung fluke mainly reported in Korea, China, Taiwan, Japan, and the Philippines.,,P. westermani worldwide.,The genus P. westermani through DNA analysis.,,,,,,P. westermani are clustered into at least two groups (East Asia and South/Southeast Asia), in association with geographically distinct distributions.,,,,Paragonimus spp. have been published so far. The only report was our previous study on ITS2 DNA sequence of ancient P. westermani eggs obtained from a 17th century Korean mummy.P. westermani was very similar to that of modern P. westermani reported in East Asia, but was genetically distinct from the P. westermani sequences of Southeast Asia.Recently, researchers have attempted to reveal the genetic characteristics of P. westermani genetic marker (COI) has been continuously accumulated by multiple researchers.,,,,Paragonimus eggs in many more coprolite specimens (n = 6) from the 15th to 18th century Korean mummies.,P. westermani eggs in order to obtain more comprehensive information about the evolutionary history of P. westermani.Nevertheless, our previous study was performed with only one sample and a single genetic marker (ITS2). Over the past several years, the genetic information of another The samples used in this study were acquired from the 15th to 18th century Joseon-era mummies . The specimens were ancient faeces from the intestines of mummies (Cheongdo and Hwasung) or the organic materials compiled upon the hipbones of half-mummified bodies .To authenticate our work on ancient DNA (aDNA), we followed the Criteria of Authentication established by Hofreiter et al.P. westermani COI and ITS2 regions were generated by Integrated DNA Technologies, Inc. . The information of the primers used in our study is summarised in For DNA extraction, we followed the method described in our previous report.TM ND-1000 Spectrophotometer . Extracted DNA (10 μL) was treated at 37ºC with 1 unit of uracil-DNA-glycosylase  for 30 min. It (40 ng) was then mixed with the reagent premix containing 10 pmol of each primer and 1X AmpliTaq Gold® 360 Master Mix . PCR conditions were as follows: pre-denaturation at 95ºC for 10 min, 45 cycles of denaturation at 95ºC for 30 s, annealing at 54-60ºC for 30 s, extension at 72ºC for 30 s, and final extension at 72ºC for 10 min. The polymerase chain reaction (PCR) products were separated on 2.5% agarose gel , and then stained with ethidium bromide. Negative controls (extraction controls) were also applied to the electrophoresis at the same time. Electrophoresis results were photographed using a Vilber Lourmat ETX-20.M equipped with Biocapt software .DNA quantification was performed using a NanoDropThe PCR amplicons were isolated using a QIAquick Gel Extraction Kit . The bacteria were transformed using the pGEM-T Easy Vector system . Transformed bacteria were grown on an agar plate containing X-GAL (40 μg/μL), ampicillin (50 μg/mL), and 0.5 mM IPTG for the 14 h. After selected colonies were grown in LB media for 12 h, the purification of cultured bacteria was performed using a QIAprep® Spin Miniprep kit .Each amplified strand was sequenced using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit  and the 3730xl Automatic Sequencer . The obtained DNA sequences were aligned by MEGA7 program.P. westermani and other taxa from NCBI GenBank was inferred from the Phylogeny Reconstruction analysis implemented in MEGA7.The evolutionary relationship of P. westermani-specific primers (COI1 and ITS2-1 in P. westermani primer sets COI1 (106 bp) and ITS2-1 (133 bp) were detected in only two samples (Cheongdo and YG2-4) while negative controls (extraction controls) did not exhibit any amplified bands ]. The consensus sequences of the P. westermani COI and ITS2 regions from the Cheongdo and YG2-4 mummies were identical [Supplementary data ].To obtain the consensus sequences of COI and ITS2 regions, we repeated cloning and sequencing for each specific amplicon. From these trials, 9-12 clone sequences were successfully acquired for the COI (390 bp) and ITS2 (417 bp) regions. Next, multiple sequence alignment was performed using Clustal W implemented in MEGA7. We then obtained consensus sequences after alignment of the cloned sequences [Supplementary data ]. The BLAST results are summarised in P. westermani COI sequences obtained from Joseon mummies exhibited significant similarities to the P. westermani COI sequences reported in Korea , Japan , China , Vietnam (FJ434988.1), India , the Philippines (U97213.1), Thailand (AB354224.1), and Sri Lanka (AY240940.1). We also found moderate similarities to the GenBank sequences of P. siamensis (AB354231.1), P. paishuihoensis (AB679289.1), and P. mexicanus (KC562301.1) .Using NCBI/BLAST tools, we compared the consensus sequences with those available in GenBank [P. westermani sequences exhibit similarities to P. westermani ITS2 sequences found in Korea (AF333277.1), Japan (U96907.1), China , India , Taiwan (U96908.1), Vietnam , Malaysia (U96909.1), Thailand , and Sri Lanka (AY240942.1). Similar sequences were also found in the sequences of P. siamensis (AB354222.1), Euparagonimus cenocopiosus (AF159601.1), and P. skrjabini (AB703444.1). The P. westermani ITS2 sequence obtained from a Korean mummy reported in our previous study ; In the case of the ITS2 region, the current Joseon P. westermani COI and ITS2 regions . P. westermani sequences were clustered into several groups. In the case of the COI region, Group I included P. westermani sequences from East Asia  whereas Group II included those from South and Southeast Asia  and formed another cluster. We note that some sequences reported from India and Sri Lanka formed a separate cluster of the third separate group (Group III)  A. P. wesa groups A.P. westermani ITS2 sequences reported from East Asia , Southeast Asia , and South Asia  were also separately clustered in the phylogenetic analysis  and Southeast Asia  were clustered with those of the East Asia group in this study (Like the COI region, analysis B. The P.is study B. SimilaP. westermani infection in the Joseon society might reach as high as 33.3%.,,,P. westermani. In this study, using coprolite samples obtained from Joseon dynasty mummies, we successfully analysed the COI and ITS2 regions of aDNA from P. westermani. As very few studies have examined ancient P. westermani DNA so far,P. westermani paleogenetics. Nevertheless, we admit that aDNA reports of much wider geo-historical scope are still required to improve our knowledge about the exact evolutionary history of P. westermani.According to the paleoparasitological estimation, the prevalence of


However, the influence of the grain size of Ba(B′1/3B″2/3)O3 ceramics with nano or sub-micron grains on the ordering domains structure is still not clear. In the present paper, highly dense (~98%) BZT microwave dielectric ceramics with homogeneous sub-micron structure (~330 nm) were prepared through spark plasma sintering (SPS). High resolution transmission electron microscopy combined with X-ray diffraction (XRD)clearly showed that the B-site ordering structure of sintered BZT samples by SPS becomes the B-site long-range 1:2 ordering as annealing proceeds. In contrast, the short-range 1:2 ordering in non-annealed counterparts was also present, which was not detectable by XRD. The size of B-site ordering domains enlarged with annealing temperature. The sub-micron structure of sintered BZT ceramics by SPS remained stable at up to 1400 C; however, the size of B-site 1:2 ordering domain was more than five times larger, which led to a significant increase of the quality factor (Q·f) to 37,700 GHz from 15,000 GHz.The degree of Zn They attributed the improved Q value to the segregation of Zr4+ ion to the boundary region to stabilize the domain boundaries.Complex perovskite BaB′/3B″2/3O3nologies . It has ordering ,7, orderordering ,10,11,12ordering ,14,15. Tf values . Furtherordering . With th1/3B″2/3)O3 perovskite ceramics is significantly affected by ordering domain boundaries and grain boundaries [Q values result from the grain growth that accompany a decrease in the amount and density of grain boundaries due to elevated annealing temperature and lengthy annealing time [5 ceramics increases with grain growing [1/3Nb2/3)O3 samples decreased with increasing grain size [Although the complex relationship between quality factor and ordering domain is to be expected, this progress is impeded by extrinsic losses including the secondary phase, porosity, impurities, oxygen vacancies, and dislocations . Additioundaries . Howevering time . Similar growing . On the ain size . Due to ain size . As a coain size ,23,24. 3-0.3NdAlO3 microwave ceramics can be fabricated by SPS technology [Q·f values of sintered BZT ceramics are closely related to the increasing ordering domain size during the annealing processing; however, a crucial issue is that the evolution of B-site 1:2 ordering degree and ordering domains needs to be clarified in this processing for polycrystalline Ba(B′1/3B″2/3)O3 ceramics with grain size falling to sub-micron level. Considering the high grain boundaries density in sub-micron structured BZT ceramics, another question that should be solved is what is the effect of grain-boundaries on the quality factor in Ba(B′1/3B″2/3)O3 ceramics with sub-micron grain size.Recently, spark plasma sintering (SPS) technique, as one of the field assisted sintering techniques, has received special attention because it can be used to effectively tailor microstructure at a relatively low temperature for a significantly decreased soaking time ,26,27,28chnology . Prelimi1/3B″2/3)O3 ceramics, the present paper aimed to investigate the evolution of B-site ordering and ordering domains in sub-micron structured BZT ceramics fabricated by SPS during the annealing processing. Special attention was paid to the relationship between the ordering domain, grain size and dielectric loss of sub-micron structured BZT perovskite microwave dielectrics.Using BZT ceramics with sub-micron structure as a prototype of Ba powders were used as raw materials. They were ball milled in ethanol at a speed of 300 rpm for 24 h. Then, the dry powders were re-milled at a speed of 500 rpm for 12 h. Then, the powder was calcined at 1350 °C for 5 h to synthesize BZT powder. Subsequently, the calcined powder was milled again with ethanol at a speed of 300 rpm for 24 h to optimize the size of powder and dried in air. In order to synthesize the nano-sized BZT powders, they were prepared by traditional route with mechanical milling processing. BaCOThere is a densification stage and a non-densification stage (grain growth and pores elimination) in the sintering processing of SPS . Our preThe relative density of the sintered BZT samples was calculated by the Archimedes’ theory. The X-ray diffraction XRD technique  was used to examine the structure and B-site 1:2 ordering. Microstructure of thermally sintered and annealed BZT ceramics was surveyed by Scanning Electron Microscope . Ordering domain structure of annealed BZT ceramics was characterized by the spherical aberration corrected Transmission Electron Microscopy  with correction value (C3) of −500 nm.rε) and quality factor (·fQ) of the samples were measured by a network analyzer  using the Hakkicoleman dielectric resonator method modified by Courtney [The dielectric constant O3 perovskite ceramics; however, its accuracy is usually restricted by scattering length and volume fraction [fraction . Our prefraction . The B-sfraction . This faIt is well-known that the ordering domains can be induced by proper annealing treatment. The B-site 1:2 ordering degree of BZT samples depends on the subsequent annealing treatment as well. The XRD patterns of the sintered BZT ceramics that are subsequently annealed at different temperature for 12 h are shown in ns peaks . In addiTEM provides a stronger tool to reveal the evolution of B-site 1:2 ordered domain and domain boundaries in BZT ceramics. TEM is especially well-suited to survey the B-site ordering of complex perovskites ,34. Sele2+ and Ta5+ ions ordering of BZT samples [High resolution transmission electron microscopy (HRTEM) was further utilized to characterize the domain of 1:2 Zn samples ,36. The In contrast, the density of ordering domains in the annealed BZT ceramics at 1300 °C significantly increased, although the size and shape of ordering domains were slightly irregular, as shown in As the temperature of annealing rose to 1400 °C, the size of the ordering domains labeled A, B, C of annealing samples continued to grow, and the domains possessed about 75% of the area of the image, as shown in 1/3B″2/3)O3 perovskite ceramics [With the growth of ordering domains, the number of domain boundaries gradually decreased. The reduction of the number of domain boundaries was be benefit to the improvement of the quality factor. It is well known that there are very high losses at the domain boundary regions due to the elastic strain, unfavorable coulombic interaction and chemical inhomogeneity in BaO3 ceramics by SPS following annealing processing increased. In contrast, the B-site 1:2 ordering structure comprising small ordering domains in non-annealed counterparts was present that was not detectable by XRD. It was observed that the grain size of sub-micron structured Ba(Zn1/3Ta2/3)O3 by SPS remained stable up to 1400°C; however, the size of B-site ordering domain was more than five times larger (from ~8 to >40 nm), which led to a significantly increase of the quality factor (·fQ) to 37,700 from 23,600 GHz. Our results show that the ordering induced domains rather than grain boundary might play a leading role in increasing the quality factor of BZT microwave ceramics in the submicron range.Highly dense (~98%) BZT microwave dielectric ceramics with homogeneous sub-micron sized grains (~330 nm) and B-site 1:2 ordering were prepared by SPS. The size of B-site 1:2 ordering domains in sintered Ba(Zn


Bacteria and Archaea. A quantitative measure of domain promiscuity, the weighted domain architecture score (WDAS), was used and applied to 1317 domains in 1320 genomes of Bacteria and Archaea. A functional analysis associated with the WDAS per genome showed that 18 of 50 functional categories were identified as significantly enriched in the promiscuous domains; in particular, small-molecule binding domains, transferases domains, DNA binding domains (transcription factors), and signal transduction domains were identified as promiscuous. In contrast, non-promiscuous domains were identified as associated with 6 of 50 functional categories, and the category Function unknown was enriched. In addition, the WDASs of 52 domains correlated with genome size, i.e., WDAS values decreased as the genome size increased, suggesting that the number of combinations at larger domains increases, including domains in the superfamilies Winged helix-turn-helix and P-loop-containing nucleoside triphosphate hydrolases. Finally, based on classification of the domains according to their ancestry, we determined that the set of 52 promiscuous domains are also ancient and abundant among all the genomes, in contrast to the non-promiscuous domains. In summary, we consider that the association between these two classes of protein domains (promiscuous and non-promiscuous) provides bacterial and archaeal cells with the ability to respond to diverse environmental challenges.In this work, we describe a systematic comparative genomic analysis of promiscuous domains in genomes of  Since Wetlaufer  describeTherefore, proteins are modular, and it has been suggested that large proteins contain multiple domains, either with the same or with different structural folds . In thisRecently, some of these domain combinations have been identified as stable during evolution, whereas others have been highly mobile . AccordiTherefore, in this work we evaluated the tendency of protein domains to combine with other domains, i.e., domain promiscuity, in proteins of bacterial and archaeal genomes. We assigned domains based on a Superfamily hidden Markov models (HMM), and a quantitative measure of domain promiscuity, the weighted domain architecture score (WDAS), was used to analyze 1319 non-redundant bacterial and archaeal genomes. We further attempted to correlate this measure with function, genome size and protein fold ancestrality.Bacteria and Archaea domains were downloaded from the NCBI server and filtered to exclude redundancy, using the criteria described in [A total of 5321 genomes corresponding to ribed in . We perfThe predicted protein sequences (the proteome) in all organisms were scrutinized to assess their domain organization by using the Superfamily database assignments . A libraPt is the total proteins per genome and Pd is the number of proteins with domain d.The WDAS, a measure of domain architecture diversity, was computed as described elsewhere . The WDAd, is:fd is the number of different domain families adjacent to domain d.To determine the diversity of architectures associated with a specific domain, the inverse variability (IV) is obtained from the inverse of the number of distinct partner domain families at the N- and C-terminal sides adjacent to a domain. The definition of the IV of a domain, Finally, the WDAS of a domain is the product of the IAF and the IV:k is the number of equal classes and N the number of data, rounding to the nearest integer, the k value. Thus, the width of classes was determined with the equation: c = R/k where R = high value–low value (Genome size in ORFs). Values resulting from the application of the above formulas were k = 13 and c = 836, thus 13 windows without overlaps were used with a width of 836 ORFs [The associations between the WDAS and genome size (measured in ORFs) were binned in 13 intervals without overlaps, with a width of 836 ORFs, as previously described . In brie836 ORFs .In order to define the threshold to separate a domain as promiscuous and non-promiscuous, we computed an accumulative plot for the number of enriched functions associated to promiscuous and non-promiscuous domains at different scores of WDASs. In a posterior step, both datasets were adjusted with polynomial regression . From thx domains associated with a particular category in a list of interest k, from a set of N domains, with m domains associated with the same category. The P-values were corrected for multiple testing with Benjamini-Hochberger method to decreases the false discovery rate, and a statistical significance at P-value of <0.05 was set.For each genome, two lists of protein domains were considered, using a threshold of ≤2.7 for promiscuous and ≥2.8 for non-promiscuous domains. An enrichment analysis using a hypergeometric test was conducted on these lists, considering 50 different functional categories obtained from the Superfamily (SUPFAM) and Structural Classification of Protein (SCOP) databases. Such a distribution describes the probability of finding Gij), average genome occurrence (Ḡi), and fraction of genomes harboring a fold (fi). Gij defines how often a protein fold (i) occurs in a given proteome (j). Ḡi represents averages of Gij values. Ḡi and fi measure the extent of fold sharing within each domain or combination of domains. Values were converted into linearly ordered multistate characters and normalized using an arbitrary scale . Cumulative frequency plots were used to illustrate the accumulation of folds belonging to a protein class along a phylogenetic tree, and it is a function of distance in nodes from the root. These plots can be considered time plots of lineages [To evaluate the protein domains ancestrality, superfamily assignments were associated to the phylogenomic tree of Protein Fold Families values previously described . In brielineages  with a tCandidatus Evansia muelleri” (GCF_000953435.1), with coverage of more than 95% and representing the organism with the highest proportion of proteins with domain assignments. This result is consistent with the assignment percentage of proteins that were assigned with at least one Superfamily domain for Eukaryotes, Archaea, Bacteria and Viruses [Mycoplasma haemofelis Ohio2 (GCF_000186985.1), a hemoplasma species with a coverage of less than 30% and M. suis str. Illinois (GCF_000179035.2), M. ovis str. Michigan (GCF_000508245.1), and Spiroplasma kunkelii CR2-3x (GCF_001274875.1), with coverages of 33.4%, 33.6%, and 38.2%, respectively, suggesting that a large diversity of novel protein domains remains to be identified in those genomes and must be further explored  between the abundance of multidomain proteins and the number of proteins per genome  and Sorangium cellulosum So0157-2 (GCF_000418325.1_ASM41832v1), among others. This finding is interesting because it shows that a low proportion of domains are associated with the highest number of architectural combinations, compared to non-promiscuous domains.To evaluate the set contribution of promiscuous and non-promiscuous domains to the total repertoire of domains associated with all the genomes, those domains with a WDAS between 0 and 3.0 See  were conBased on the definition of promiscuity, it was found that the proportion of promiscuous domains represents less than 10% of all non-redundant domains per genome, suggesting that a small fraction of the total repertoire of domains is determinant for the diversity of combinations of architectural identified so far. Therefore, in order to determine the preferential functions of promiscuous and non-promiscuos domains per genome, both datasets were evaluated, considering the functional annotations deposited in the SUPFAM and SCOP databases. In these databases, 50 different functional groups have been determined, and they are classified in eight broad categories . From this analysis, 18 functional categories were identified as significantly enriched in the promiscuous domains in at least one genome, and in particular the Small-molecule binding domains were identified as enriched in 1219 genomes, whereas Signal transduction was enriched in 377 genomes and DNA-binding (transcription) and transferases were enriched in 153 and 118 genomes, respectively. Finally, the other 14 functional categories identified as enriched in minor proportions, such as Polysaccharide metabolism and Inorganic ion transport and metabolism and, were associated with 34 and 25 genomes, respectively .In contrast, non-promiscuous domains were associated with 6 functional categories, with Unknown function, Translation, ribosomes, ribosome biogenesis; tRNA metabolism and DNA replication, recombination, repair, the most enriched. These functions, associated to the non-promiscuous domains were identified in 207, 112 and 8 genomes, respectively. Therefore, functional associations were different between promiscuous and non-promiscuous domains, suggesting that bias to DNA binding, Transferases, Small-molecule binding and Signal transduction functions are specifically associated with promiscuous domains. In our second approach, we considered the domains as a function of their correlation to genome size, where the correlation between a WDAS for a given domain and the genome size (number of ORFs) could be positive or negative and a set of uncorrelated domains could be identified. To this end, R-values were calculated and two large groups were defined: those negatively correlated and those with no correlation. Therefore, we were interested in the set of negatively correlated domains , because they showed WDAS values decreasing as a function of genome size, i.e., protein domains are less promiscuous in small genomes and they are more promiscuous in large genomes. In addition, only protein domains identified in more than 60% of the total genomes or 770 different genomes were considered in this analysis, because we were interested in those domains with a wide genomic distribution for our analysis See .From the complete set of 1317 protein domains analyzed, 52 protein domains had negative correlation values , i.e., the number of architectures associated with them increased as a function of genome size . In thisIn summary, the four functional categories more abundant associated to this set of promiscuous domains are related to metabolism , DNA-binding and small-molecule binding. These functional categories suggest that these structural domains could be associated with ancient and global functions that define the basic functions of cellular maintenance, signaling and regulation of gene expression, and they are influenced by the dynamics of the genome architecture in bacteria ; i.e. anAlthough there are diverse and interesting protein superfamily domains, there are two cases to illustrate the association of promiscuity with genome size: the P-loop–containing nucleoside triphosphate hydrolases domain  and the Winged helix-turn-helix domain (SSF:46785).2+ cation, respectively [The P-loop domain is the most prevalent domain of the nucleotide-binding protein folds, has been classified in the small-molecule binding functional category (HA) and represents 13.8% of the total domains identified as promiscuous. Proteins with this fold catalyze the hydrolysis of the beta-gamma phosphate bond of nucleoside triphosphate (NTP), and the energy from NTP hydrolysis is used to induce conformational changes in other molecules . P-loop ectively . Thus, wBacteria to Archaea, and includes diverse families, such as the Catabolite gene activator (CAP) family, in which global regulators (CRP and FNR) have been described in the bacterium Escherichia coli [A second representative example is associated with the Winged helix-turn-helix DNA-binding domain. These structures are characterized by the presence of a third alpha-helix and an adjacent beta-sheet and are central components for DNA binding; the “wings” are small beta-sheets . The rechia coli , the heahia coli . The WDAIn summary, we suggest that the set of 52 superfamilies follows trends similar to those of the P-loop and wHTH superfamilies, reinforcing the notion that these superfamilies not only increase their associations with other domains but also their architecture diversity.To evaluate how promiscuity developed during evolution, we mapped the WDASs associated with all domains against the ancient approach described by . This anZ-Score is -10.62215 with a p-value of < 0.00001, being significant at p < .01. In other words, the difference between the promiscuous and non-promiscuous datasets is statistically significant. In summary, the promiscuous domains are abundant and ancient, such as the P-loop–containing nucleoside triphosphate hydrolases (SF:52540) and the DNA-binding domain (wHTH), because they are recurrent and most ancestral among the universal domains, in comparison to non-promiscuous domains. Therefore, the interplay of promiscuous and no-promiscuous domains determines the architecture of proteins in all the genomes.In In this work, we evaluated 1317 superfamilies for their architectures, their abundance in bacterial and archaeal genomes, their functional roles and their antiquities. Our data reinforce that increased gene complexity also requires the development of mechanisms for gene regulation at the transcription level and small-molecule binding, where the P-loop and the wHTH are the most significant examples, suggesting that the interplay of these structural domains could increase the ability of an organism to recognize and respond to diverse environmental stimuli. These findings are relevant in the context of functional categories, because promiscuous domains are mainly associated with basic metabolic, signal transduction, and gene regulation functions. We also found 52 domains whose promiscuity value or WDAS depends on genome size, and this was reinforced by the number of domains associated with each of them. In summary, we consider the association between these two classes of protein domains (promiscuous and non-promiscuous) has provided, since ancestral times, bacterial and archaeal cells with the ability to respond to diverse environmental challenges.S1 Fig2. A polynomial regression of grade 4 was found as the best for both datasets.The distributions were adjusted using the function polyfit of the numpy package in python, and different degree of polynomial regression were tested to maximize R(TIFF)Click here for additional data file.S2 FigOn the X-axis is the number of ORFs per genome, and the Y-axis shows the total of proteins with two or more domains. Each open circle denotes a genome. . A non-linear least squared to fit the dataset was used.(TIFF)Click here for additional data file.S1 TableColumns correspond to: Genome ID; Total of Proteins per genome (ORFs); NRProteins_Assigned ; Coefficient ; NRDomains; 1Domain_count; 2Domain_Count; 3Domain_Count; > = 4DomainCounter.(TXT)Click here for additional data file.S2 TableColumns correspond to: Domain; C.C.; p-value, Gen_Count; Domain_Count Function; Group_Count; Firmicutes; Proteobacteria; Euryarchaeota; Actinobacteria; Tenericutes; Chlamydiae: Chlorobi; Crenarchaeota; Fusobacteria; Bacteroidetes; Spirochaetes; Cyanobacteria; Deinococcus; Thermus; Thermotogae: Aquificae; Chloroflexi; Gemmatimonadetes: Deferribacteres; Acidobacteria; Candidatus Korarchaeota; Verrucomicrobia: Elusimicrobia; Dictyoglomi; Nitrospirae; Fibrobacteres; Synergistetes; Thermobaculum; Planctomycetes; Cloacimonetes; Chrysiogenetes: Thermodesulfobacteria; Ignavibacteriae; Caldiserica; Thaumarchaeota: Armatimonadetes; Taxonomy total distribution.(TXT)Click here for additional data file.S3 TableColumns correspond to: Domain; Function; SCOP; Description; ancient_order; Ancient_score; R-value; Total_Domains; Archaea_Domains; and Bacteria_Domains.(TXT)Click here for additional data file.S1 FileOn the X-axis of each graph, genome size ranges are displayed in 13 windows, with a range of 836 ORFs each. On the Y-axis are the WDASs. The lines shown in the boxes are the median values. The whisker caps represent the minimum and maximum values. Superfamily IDs correspond to the names in (ZIP)Click here for additional data file.