Within corals, superoxide may contribute to pathogen resistance but also bleaching, the loss of essential algal symbionts. Yet, the role of superoxide in coral health and physiology is not completely understood owing to a lack of direct in situ observations. By conducting field measurements of superoxide produced by corals during a bleaching event, we show substantial species-specific variation in external superoxide levels, which reflect the balance of production and degradation processes. Extracellular superoxide concentrations are independent of light, algal symbiont abundance and bleaching status, but depend on coral species and bacterial community composition. Furthermore, coral-derived superoxide concentrations ranged from levels below bulk seawater up to ∼120 nM, some of the highest superoxide concentrations observed in marine systems. Overall, these results unveil the ability of corals and/or their microbiomes to regulate superoxide in their immediate surroundings, which suggests species-specific roles of superoxide in coral health and physiology.The reactive oxygen species superoxide  that can influence coral health. Diaz and colleagues conduct  Coral reefs are among the most biologically rich and economically valuable ecosystems on the planet13Symbiodinium—essential algal endosymbionts that provide colour, organic carbon and nutrients to the coral host4Symbiodinium to tolerate elevated temperatures10Coral bleaching involves the loss of 2·−). During bleaching, light and heat stress damage the photosynthetic machinery of Symbiodinium cells and impair mitochondrial electron transport in the coral host, which is thought to result in the over production of intracellular ROS, the onset of oxidative stress and an antioxidant response throughout the coral holobiont512Roseobacter, Vibrio and other genera commonly found in coral microbiomesSymbiodinium isolates representing clades A and C produce extracellular superoxide even in the absence of heat and light stress, potentially via transmembrane oxidoreductases, such as NADPH oxidaseStylophora pistillata in aquaria incubations under non-stressful conditionsReactive oxygen species (ROS) play a critical yet enigmatic role in coral bleaching and health. ROS include intermediates in the reduction of molecular oxygen to water, such as the superoxide radical anion H oxidoreductases putatively involved in the production of extracellular superoxide are associated with increased thermotolerance of the coral in vivo measurements of ROS difficult. To advance our understanding of superoxide dynamics in the coral holobiont, we capitalized on recent advances in non-invasive chemiluminescent techniques to make the first in situ measurements of external superoxide production by several species of thermally stressed and bleaching corals in a natural reef environment.Both intracellular and extracellular superoxide production are clearly important to maintaining the redox homoeostasis and health of corals. Despite the vast array of possible superoxide sources identified in corals, however, the actual origins, distributions and ecological underpinnings of superoxide production in natural coral communities remain largely unknown due to a lack of direct superoxide measurements. Indirect evidence of oxidative stress in bleaching corals is based on observations of antioxidant activity, gene expression and proteomic profiles, yet the methodologies available for directly measuring intracellular ROS are invasive and artifact prone3233Fungia scutaria, Montipora capitata, Pocillopora damicornis, Porites compressa and Porites lobata. Superoxide concentrations at coral surfaces could not be explained by abiotic photo-driven mechanisms of ROS production, photosynthesis, bleaching status or Symbiodinium abundances. Extracellular superoxide production by bacterial symbionts and asymbiotic coral larvae was confirmed in laboratory experiments, supporting the conclusion that superoxide production at the coral surface may originate from the activity of epibionts or the coral host itself.The goal of this study was to determine whether and to what degree various coral species produce external superoxide on a natural reef and to assess the potential role of coral symbionts in this superoxide production. Results revealed significant species-level control of external superoxide concentrations by the corals M. capitata) to steady-state concentrations that were ∼120 nM higher than bulk seawater (P. lobata)  . These s nM ref.  and from nM ref. , respectF4,81=34.8, P<0.01; F1,81=1.27, P=0.26) or when health was separately compared for each species , F. scutaria and P. damicornis (P=0.83) and M. capitata and P. damicornis  on superoxide concentrations. This analysis revealed that average coral-derived superoxide levels were significantly different as a function of coral species , suggesting that corals are a point source, consistent with the short lifetime of superoxide in these waters  . Second,The potential direct and indirect pathways of external superoxide formation by corals are currently unresolved. In addition to superoxide, corals also release dissolved organic carbon424446Porites sp. (∼500 nM), intermediate for Pocillopora sp. (∼250 nM) and near zero for Fungia sp.184044M. capitata were lower than the surrounding seawater. Moreover, the presence of stable, non-zero levels of superoxide and hydrogen peroxide at the surfaces of coral species that have the ability to degrade external ROS indicates that these external ROS concentrations are not detrimental. While superoxide levels as low as 1 nM can inactivate certain enzymesThe species-level trends observed in average superoxide levels are in line with previous measurements of the ROS hydrogen peroxide, which also demonstrated a broad range of species-specific levels at coral surfacesSymbiodinium cells inhabit the symbiosome inside the host's gastrodermal tissue beneath the coral's outermost  cell layer. Despite recent evidence that the symbiosome has a pH of ∼4  or separately   , which pSymbiodinium cells are still undoubtedly a source of internal superoxide to their coral hosts , and minimal archaeal sequences. In addition, members of Verrucomicrobia and Planctomycetes were prominently associated with M. capitata and P. damicornis  but not health state (pigmented versus bleached)  , except for P. damicornis and M. capitata , which exhibited the most similar external superoxide levels among the species examined for microbial community composition  . Pairwisposition . This coin vitro. The majority of coral species examined in Kaneohe Bay vertically inherit Symbiodinium from maternal colonies, precluding the ability to collect and interrogate symbiont-free larvae. Therefore, to assess the coral host's ability to produce extracellular superoxide in the absence of Symbiodinium or bacterial symbionts, gametes were obtained from broadcast spawning corals on a reef in Curaçao. Following fertilization and rearing in sterile seawater (at least 24 h of development), asymbiotic larvae of the corals Orbicella faveolata, Diploria labyrinthiformis and Colpophyllia natans produced extracellular superoxide at considerably high rates (260–821 fmol larva−1 h−1)  coral larvae and coral-derived bacterial symbionts a−1 h−1) . Disruptin vitro. A wide phylogenetic and ecological diversity of heterotrophic bacteria, including genera commonly found in corals, were recently shown to produce extracellular superoxideEndozoicomonas, which was a prevalent organism in the Porites spp. microbiomes studied in Kaneohe Bay. We tested other common coral bacteria, cultured from corals in Micronesia, which also produced substantial extracellular superoxide, including Ruegeria sp. (isolate WHOIMSCC16 from S. pistillata) and Vibrio sp. . Extracellular superoxide production by these coral-derived bacteria ranged from 0.13±0.06 to 2.2±1.2 amol cell−1 h−1  as well as bleached and pigmented colonies of S. pistillataElevated ROS levels are commonly assumed to play an antagonistic role in corals , but ROS may also be beneficial to coral physiology. In fact, healthy corals, ive CCMP 66 as welCaenorhabditis elegans protect their host from parasites by generating superoxidePorites spp., which produced the highest external superoxide concentrations, are among the most resilient species to thermal bleaching, while Montipora spp., which had the lowest superoxide levels, are more susceptibleThe potential beneficial roles of extracellular ROS in coral function and health are likely diverse and vary in the presence and absence of external stressors. For example, according to a recent study, corals may release external hydrogen peroxide to facilitate feeding on zooplankton or as a mode of defence against pathogens regulated by physical and chemical stimuli19Given the light-independent and species-specific control of superoxide levels revealed here, coupled with similar findings for aquaria-hosted corals grown under non-stressful conditions refs , 40, it In situ superoxide measurements were conducted between 10:00 and 15:00 hours on pigmented and bleached colonies of Porites compressa, Porites lobata, Montipora capitata, Pocillopora damicornis and Fungia scutaria, at six different reef sites in Kaneohe Bay, Hawaii including sites A , B , C , D , E  and F  (F. scutaria (due to lack of a permit) under the State of Hawaii Department of Land and Natural Resources Special Activity Permit #2015-49 using a hammer and chisel, placed on ice for no more than 3 h, and frozen to −80 °C. Additionally, diel superoxide measurements were conducted on adjacent colonies of Porites compressa located off the Point of Coconut Island (Site P), . We were not permitted to remove tissue from colonies at this site. For diel measurements, photosynthetically active radiation (PAR) was measured using an underwater sensor .8002° W) . Small tCypridina luciferin analogue . The FeLume system is composed of two separate fluid lines, one of which is dedicated to the analyte solution and the other to the MCLA reagent. The reagent and, as indicated, the analyte solutions, are amended with 50 μM diethylene-triaminepentaacetic acid (DTPA) to sequester trace metal contaminants that would otherwise significantly reduce the lifetime of superoxide. To measure superoxide, both the analyte solution and the MCLA reagent are independently flushed through the FeLume system at an identical flow rate using a peristaltic pump. The MCLA reagent consisted of 4.0 μM MCLA  via the specific reaction between superoxide and the chemiluminescent probe methyl in situ superoxide measurements, the FeLume was hosted onboard a small boat. To eliminate abiotic photochemical processes that may produce superoxide during in situ measurements, opaque tubing was used, and the entire analytical system was shielded from light. The MCLA reagent was kept on ice during all field analyses, which greatly improved the stability of measurements. Seawater was directly pumped into the FeLume by placing the analyte tubing at discrete positions millimetres above the coral surface at specific points of interest, as indicated in the main text and figure legends, or in the surrounding seawater with the help of a snorkeller. To minimize the length of tubing (travel time) required to transport the water from the coral surface to the instrument, the boat was positioned adjacent to the coral being measured . Flow rates were consistent within FeLume runs and varied from 6 to 8 ml min−1 between runs.For in situ superoxide measurements on the reef, we first ran a reagent blank on the boat to account for superoxide generated from the autooxidation of MCLAin situ temperature by suspending it in a bottle in the water alongside of the boat. By doing this, slight increases in the baseline signal with increasing in situ temperature as observed previously−1, final) to an aliquot of water taken from the coral surface to confirm that superoxide was responsible for the signal observed. SOD routinely lowers seawater baseline signals, reflecting the non-zero concentration of superoxide in the seawater (seawater blank), as well as the reagent blank. The coral-derived signal was obtained by subtracting the seawater signal obtained at 5–15 cm away from the coral surface from the coral surface signal, which removes both the seawater blank and reagent blank. Coral-derived superoxide concentrations were not corrected for superoxide decay during sampling and thus represent conservative estimates. The surface and coral-depth background seawater signals were corrected by subtracting the AFRW+DTPA baseline .For 2). The calibrations were conducted in the lab using the same tubing, flow rate and temperature as the in situ measurements. Considering the short lifetime of superoxide, standards were prepared immediately before analysis. Primary stock solutions were made by dissolving a small quantity of KO2 in a basic matrix . Superoxide concentrations in primary standards were quantified by measuring the difference in absorbance at 240 nm before and after the addition of SOD  and then converting to molar units based on the molar absorptivity of superoxide Corrected signals were converted to superoxide concentrations via calibration with multi-point standard curves using the superoxide source potassium dioxide  to confirm that the signal was attributable to superoxide. In all cases, the chemiluminescence signal decreased rapidly to or slightly below AFRW+DTPA baseline levels after the addition of SOD. Chemiluminescence signals collected during the decay of superoxide were extrapolated backwards in time (0.28–63 s) to the point when the primary standard was quantified. Extrapolations assumed first-order decay kinetics because decay data were log-linear.For each calibration point, a separate FeLume run was conducted as follows: first, a blank AFRW+DTPA solution was allowed to react with the MCLA reagent until a stable baseline (<4% coefficient of variation) was achieved for ∼1 min. Then the secondary standards were pumped directly into the FeLume, and the decay of superoxide was monitored for at least 1 min. Finally, SOD was added to the secondary standard (∼0.8 U mlR>0.93), with a sensitivity that ranged from 0.1 to 0.4 luminescence units per pM superoxide. The half-life of superoxide in the calibration matrices ranged from 0.41 to 0.56 min. These half-lives represent maximum estimates of the lifetime of superoxide on the reef because they were derived from AFRW amended with DTPA, which sequesters trace metals that would otherwise significantly reduce the lifetime of superoxide. The detection limit of this method, calculated as three times the s.d. of a series of blank measurements, was 0.24 nM. Given the sensitivity of superoxide to trace metal contamination, all vials and glassware were pre-cleaned with 10% HCl and washed with ultrapure (18 MΩ) water before use. All reagents were trace metal grade.Calibration curves were constructed on the basis of the linear regression of multiple standard points (extrapolated luminescence versus superoxide concentration). Calibrations yielded linear curves  or using aluminium foil (other colonies)Symbiodinium counts on the same specimen typically agreed within ±5% (s.d.).Frozen coral tissues were defrosted on ice and tissue was removed from the skeleton using an airbrush with 0.2 μm filtered seawater, blended for 30 s and centrifuged at 5,000 r.p.m. for 5 min. The supernatant was discarded, and the algal pellet was resuspended in filtered seawater and repeatedly vortexed and pelleted until the symbiont cells were free of host material. The algal pellet was finally preserved in 4% paraformaldehyde with filtered seawater and stored at 4 °C. For quantification, cells were concentrated onto 5 μm membrane filters (Millipore), mounted onto slides and imaged using Cy5 and TL DIC channels simultaneously with × 20 objective and × 1.6 optovar with a Zeiss Axio Observer.Z1 microscope and AxioCam MRm Rev.3w camera (Carl Zeiss Inc). Each image contained 1,388 × 1,040 pixels, with each pixel sized 0.32 × 0.32 μm, relevant to a 447.63 × 335.40 μm area. For each sample, 12 images from 12 fields of view under the same imaging conditions were captured with Zen 2011 software , and cell counts were then automated using a custom Matlab script and normalized to skeletal surface area, which was obtained from either a cork borer diameter (for −1 at 60 °C for 30 min). DNA was quantified with the Qubit HS dsDNA fluorescent assay (Invitrogen), and samples were shipped to the University of Illinois for amplification and sequencing. A mastermix for each sample was prepared using the Roche High Fidelity Fast Start Kit and 20 × Access Array loading reagent. Mastermix was aliquoted to 48 wells of a PCR plate. To each well, 1 μl DNA and 1 μl Fluidigm Illumina linkers with unique barcode were added. Primer sequences for respective variable regionsSamples of coral tissue were collected after the superoxide measurements using a hammer and chisel and were placed into whirl-pack bags underwater. Samples were kept on ice until arriving back at the laboratory, where they were wrapped in aluminium foil and frozen to −80 °C until processing. Coral mucus and tissue were airbrushed from the coral tissue as previously describedEndozoicomonas montiporae (LMG 24815) was obtained from the Belgian Coordinated Collections of Microorganisms. E. montiporae was grown in liquid marine broth at 23 °C. Ruegeria s.p. (WHOIMSCC16) was obtained from Stylophora pistillata and Vibrio s.p. (WHOIMSCC50) from Porites lobata, both isolated in the Federated States of Micronesia, and were grown using dilute nutrient media . Growth of all bacteria was quantified with cell counts after staining with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI).Orbicella faveolata, Colpophyllia natans and Diploria labyrinthiformis were collected in the evening 6–12 days following the full moon at the Water Factory site in Curaçao  using standard collection nets. The gametes were pooled and fertilized for ∼30 min, initially diluted into 0.45 μm filtered seawater and subsequently maintained in 0.2 μm filtered seawater with daily water changes once they developed into larvae.Egg and sperm from at least three colonies of −1) across a sterile syringe filter placed in the FeLume's analyte line for ∼2 min to generate stable baseline signals (<4% coefficient of variation). For larvae, the carrier solution consisted of AFRW amended with 75 μM DTPA (AFRW+DTPA). For bacteria, the carrier solution was 20 mM phosphate-buffered (pH=7.6) artificial seawater  amended with 75 μM DTPA (PBASW+DTPA). Next, the pump was stopped, the syringe filter was removed and using a syringe, specimens were gently deposited on the filter . Then the specimen-loaded filter was placed back inline, and the pump was restarted (2 ml min−1). In principle, superoxide produced extracellularly is entrained by the carrier solution and detected upon mixing with MCLA in the flow cell downstream of the specimens. Biological signals were collected until a stable, steady-state reading was achieved (<∼4% coefficient of variation) and maintained for at least 1 min. Finally, SOD was added to the carrier solution  to confirm that superoxide was responsible for the signal observed.Extracellular superoxide produced by laboratory cultures and coral larvae was measured using a previously described MCLA/FeLume method2 standard curves under identical conditions as biological experiments, similarly to the protocol described elsewherein situ superoxide measurements. Under the conditions used for these experiments , steady-state superoxide concentrations ranged from 0.22 to 12 nM. The typical detection limit  was 0.24±0.11 nM (avg±s.d.). As determined from calibration experiments, superoxide half-lives varied inversely with superoxide concentration and ranged from 0.7 to 3.3 min (AFRW+DTPA in Curaçao) and 4.9 to 24.8 min (PBASW+DTPA). Some half-lives in PBASW+DTPA are relatively high compared with most natural waters, but similar to superoxide decay rates measured in water samples from the Costa Rica Dome that were also filtered and amended with DTPAStable biological signals were averaged and corrected for background luminescence by subtracting the average initial baseline . Corrected biological signals were converted to superoxide concentrations via calibration with multi-point KOR>0.97 for Curaçao AFRW+DTPA; R>0.98 for PBASW+DTPA), with a sensitivity that averaged 2.2±0.6 pM per luminescence count. Net superoxide production rates were calculated as the product of the steady-state superoxide concentration and flow rate . The production rate of superoxide by each replicate was normalized to the total number of cells or larvae loaded on the filter .Extrapolation times were typically 64.6±10.6 s (avg±s.d.) under our experimental conditions. Biogenic superoxide concentrations were not corrected for superoxide decay and thus represent conservative estimates. As above, calibrations yielded highly linear curves .Superoxide data and e levels  were exaompressa . Speciescolonies  were per species , superox species  and supe species  were alsFull-length SSU rRNA gene sequences of the coral bacterial cultures are available in GenBank under accession numbers KT957318 and KT957319. Amplicon sequence data are available in as NCBI bioproject PRJNA324813. Additional data may be available from the authors upon request.How to cite this article: Diaz, J. M. et al. Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event. Nat. Commun.7, 13801 doi: 10.1038/ncomms13801 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-3 and Supplementary Tables 1-5


NBPF) was applied to this gene family when the first identified member of the family was found to be interrupted in an individual with neuroblastoma.We are jointly proposing a new name for a protein domain of approximately 65 amino acids that has been previously termed NBPF or DUF1220. Our two labs independently reported the initial studies of this domain, which is encoded almost entirely within a single gene family. The name Neuroblastoma Breakpoint Family , and the three-domain block that expanded dramatically in the human lineage will be termed the Olduvai triplet. We believe that additional studies of this domain, primarily from our laboratories over the past 10 years, have resulted in furthering our understanding of these sequences to the point where proposing a new name for this domain is warranted. Because of considerable data linking the domain to human-specific evolution, brain expansion and cognition, we believe a name reflecting these findings would be appropriate. With this in mind, we have chosen to name the domain (and the repeat that encodes it) Olduvai. The gene family will remain as The new name refers to Olduvai Gorge, which is a site in East Africa that has been the source of major anthropological discoveries in the early-mid 1900’s. We also chose the name as a tribute to the scientists who made important contributions to the early studies of human origins and our African genesis. Here we jointly propose a new name for a protein domain of approximately 65 amino acids that has been previously termed NBPF3 or DUF12204. Our two labs independently reported the initial studies of this domain, which is encoded almost entirely within a single gene family. The name Neuroblastoma Breakpoint Family (NBPF) was applied to this gene family when the first identified member of the family was found to be interrupted in an individual with neuroblastoma5. Prior to this discovery, the Pfam database had termed the domain DUF1220, denoting it as one of many proteindomains ofunknownfunction6. It has been Pfam’s intention to use “DUF” nomenclature to serve only as a temporary placeholder until more appropriate names are proposed based on research findings. We believe that additional studies of this domain, primarily from our laboratories over the past 10 years, have resulted in furthering our understanding of these sequences to the point where proposing a new name for this domain is warranted.Protein domains are portable units within proteins that can serve important biological functions. They have been implicated in a broad range of key biological phenomena, including development, disease and evolutionKey findings relevant to assigning a name to the domain are as follows:1. The domain has been repeatedly linked with human-specific evolution. The haploid human genome is estimated to encode approximately 300 copies of the NBPF/DUF1220 domain, while the copy number for other species is substantially lower: great apes 90–120, monkeys 30–40, and all other mammalian species 1–98. The increase in humans  represents the largest human lineage-specific copy number increase of any coding region in the genome. These findings, involving the copy number of a protein coding domain, provide strong support for an involvement in human-specific evolution.2. The domain has been linked with human brain evolution and cognitive function. Over the past 10 years we have published several papers on NBPF/DUF1220 protein domains and theNBPF gene family. These have implicated the copy number of the domain in human brain evolution11, brain size-related phenotypes11, brain disorders 14, and measures of cognitive function15. Also, our finding of a robust linear association between NBPF/DUF1220 copy number and brain size across primate species was confirmed by an independent study16. 17. Deposits at the gorge are estimated to cover a time span from 2.1 million to 15,000 years ago, and the fossil remains that have been identified there are thought to represent more than 60 hominins (members of the human lineage). These findings are believed to constitute the most continuous known record of human evolution over the past 2 million years, and the longest known archaeological record of the development of stone-tool industries. Olduvai Gorge was designated part of a UNESCO World Heritage site in 197918. Just as the protein domain appears to be important to human-specific evolution, so too, Olduvai Gorge has provided key insights into human’s evolutionary origin. We believe both are central to the story of what made us human.Given the above research findings, a new name for this protein domain that is related to human-specific evolution would be appropriate. We believe a name that would do this is “Olduvai” (ohl’-du-vi) (when necessary it can be abbreviated as “ODV”). This name refers to Olduvai Gorge, which is located in the rift valley of Eastern Africa. Olduvai has been the site of key paleoanthropological discoveries related to human origins and has been called “the Cradle of Mankind”, and “the Grand Canyon of Human Evolution”th century that stimulated further research into human origins and our African genesis. While we are aware that naming a protein domain after a geographic location is unusual, we believe the name Olduvai is more than a place. Rather it has come to be thought of as a symbol of the efforts of our species to understand itself.Finally, we have also chosen this name because it reflects an appreciation for the important contributions of the scientists who made major anthropological discoveries in Africa in the early/mid-20NBPF. It is also worth noting that there is an Olduvai domain that is separate from those encoded byNBPF genes: a single, likely ancestral, copy of the domain is found within thePDE4DIP/myomegalin gene. In summary, the NBPF domain and DUF1220 domain will be termed the Olduvai domain, and the NBPF repeat and DUF1220 repeat will be termed the Olduvai repeat. The primary gene family that encodes these sequences will continue to be calledNBPF, and the primary domain subtypes will retain established nomenclature 8. The three-domain block, composed of HLS1, HLS2 and HLS3 subtypes, that is tandemly repeated within several humanNBPF genes and responsible for the great majority of additional human copies of the domain, will be called the Olduvai triplet.While we believe that the domain and repeat should now be called “Olduvai”, we also propose that, for now, the gene family name should remainThe Olduvai domain hyper-amplification in the human lineage was one of the most extreme and rapid copy number expansions in the human genome, and we look forward to additional studies that may provide further insights into the role this protein domain family plays in human disease and evolution. The authors have taken into account the reviewers remarks. I hope that this paper will encourage the NBPF and myomegalin communities to join efforts to unravel the function of the Olduvai domain and its role in the emergence of mankind.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I approve the new version of the manuscript.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This brief opinion article describes the authors' proposal to rename a domain found in a discrete group of proteins, which was originally named DUF1220 (domain of unknown function 1220) and subsequently reassigned as NBPF. The NBPF nomenclature referenced the fact that this domain is found in proteins encoded by a family of genes initially reported as the "Neuroblastoma BreakPoint Family"; however, as human gene nomenclature tries to move away from directly referencing specific phenotypes and conditions, the reference to neuroblastoma has been removed from the names for the NBPF genes, and they are now simply the "NBPF family". Interestingly, as well as this family the only other protein this domain is found in is the large "myomegalin" protein in mammals, encoded by the PDE4DIP gene. I think the fact that this is not mentioned anywhere in the article is perhaps an oversight on the part of the authors which could be remedied. It should be noted that the authors have already contacted PFAM to request this change and HGNC were also consulted and agreed this update would not create problems from a gene naming perspective. As we said then, we think it would be more informative for the new domain name to reflect functional information as opposed to something more esoteric, but the authors are not advocating any parallel change in the NBPF gene family nomenclature which is quite widely used for this family. A parallel change to the gene names would perhaps be more difficult to justify. Therefore we told the authors that we see no issue with their proposal. Importantly, this article represents the collaborative effort of two independent groups, who easily could be viewed as "rivals". Therefore this article effectively shows that such collaborations are possible and beneficial to the scientific community at large; this kind of activity should be encouraged wherever possible and I applaud the authors for making the effort to work productively together.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.  (PDE4DIP), neither of the current names, NBPF or DUF1220, is commonly used by the myomegalin community. In this respect, I think that a paragraph presenting the myomegalin family and discussing the presence of a single Olduvai “repeat” amongst its members should be added prior to indexing.This paper proposes to change the name of a protein domain from NBPF (Neuroblastoma Breakpoint Family) to Olduvai, arguing that this new name illustrates the fact that the domain copy number has been linked with human evolution. Although, it is in principle not recommended to multiply the names for a domain, the fact that the change is submitted by two major players in the field makes the proposal acceptable as it should rapidly spread in future works on this promising domain. In addition, the limited distribution of the domain should facilitate its diffusion. However, it should be noted that although having a very limited distribution found in roughly two protein families, i.e. NBPF and myomegalin The previous name, NBPF, was derived from a breakpoint in the first member identified and was also applied to the protein family. In the future, the use of the new name Olduvai only for the repeat will avoid the confusion between NBPF designating both the protein family and the repeat.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This manuscript describes a straightforward proposal to rename the NBPF/DUF1220 domain to the Olduvai domain. This is welcome because the domain has been referred to by two different names in the past.  The authors are the major players in the field, who have used the divergent nomenclature and thus will be able to ensure that the proposed naming does become the standard.  The authors had already contacted me to ask about having the domain renamed within the Pfam database, which has now been done .  Perhaps calling this paper a proposal is a bit weak. The authors should consider renaming the article to “Changing the name of the NBPF/DUF1220 domain to the Olduvai domain”. There are no agreed upon standard naming schemes for protein domains or families. To date the most popular schemes are based on naming after one or more proteins that contain the domain, sometimes based upon the initial letters of these members.  A few domains are named after scientists.  I don’t recall any domains or families named after places.  However, the renaming makes sense within the context of the evolution of this domain and human evolution. I think it would be worth mentioning the reasons that the NBPF nomenclature is retained for the gene family. I understand this is to fit with the HGNC guidelines.Minor points Please change PFAM to Pfam throughout the text.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


SLC4 family. A novel conserved Zn2+-binding motif present in the N-terminal domain of NDCBE is identified and characterized in vitro. Cellular studies confirm the Zn2+ dependent transport of two electroneutral bicarbonate transporters, NCBE and NBCn1. The Zn2+ site is mapped to a cluster of histidines close to the conserved ETARWLKFEE motif and likely plays a role in the regulation of this important motif. The combined structural and bioinformatics analysis provides a model that predicts with additional confidence the physiologically relevant interface between the cytoplasmic domain and the transmembrane domain.The sodium-driven chloride/bicarbonate exchanger (NDCBE) is essential for maintaining homeostatic pH in neurons. The crystal structure at 2.8 Å resolution of the regulatory N-terminal domain of human NDCBE represents the first crystal structure of an electroneutral sodium-bicarbonate cotransporter. The crystal structure forms an equivalent dimeric interface as observed for the cytoplasmic domain of Band 3, and thus establishes that the consensus motif VTVLP is the key minimal dimerization motif. The VTVLP motif is highly conserved and likely to be the physiologically relevant interface for all other members of the  The sodium-driven chloride/bicarbonate exchanger (NDCBE) is a member of the sodium-bicarbonate cotransporters (NBCs) within the solute carrier 4 (SLC4) gene family2. The NBCs include, among others, the electroneutral sodium-bicarbonate cotransporter (NCBE/NBCn2), which is mainly expressed in the brain especially in the choroid plexus3, and the electrogenic sodium-bicarbonate cotransporter 2 (NBCe2), which is mainly expressed in the kidney, liver and the choroid plexus4. NDCBE is electroneutral and believed to exchange one Cl− for two HCO3− and one Na+ 5. In the brain, NDCBE is primarily associated with the presynaptic vesicles of glutamatergic and GABAergic neurons6. Even though no human disease has been associated with NDCBE mutations, knockout mice exhibit defective regulation of Na+ reabsorption and reduced neuronal excitability8. In addition, low NDCBE expression levels have been reported in mouse brains in connection with chronic continuous hypoxia9. The primary structure of SLC4 transporters is divided into three major domains, an N-terminal regulatory domain, a transmembrane domain containing 14 membrane-spanning helices, and a C-terminal domain often associated with binding to carbonic anhydrase11. The transmembrane domain is largely conserved across SLC4 family members and shares more than 38% sequence identity with anion exchangers (AEs) and NBCs2. In the plasma membrane, most SLC4 transporters are believed to form functional dimers. The molecular basis for the dimerization was recently confirmed by a crystal structure of the transmembrane dimer of AE1 (PDB ID: 4YZF) reported by Arakawa et al. at 3.5 Å resolution10. The crystal structure did not include the cytoplasmic regulatory domains, and the organization of these with respect to the transmembrane dimer is still based on models. The primary structure of the isolated cytoplasmic regulatory domains shares lower sequence identity across the SLC4 family, than the transmembrane regions. Based on sequences with either low or high sequence variability, the cytoplasmic domains have been divided into regions referred to as constant regions (CRs) and variable regions (VRs)12. The VRs mainly include the different functional and regulatory motifs of SLC4 family members and their isoforms13. In addition, the VRs have been classified as intrinsically disordered regions, which are a specific dynamic type of element that lack any ordered secondary structures such as helices or strands15. The crystal structure of the cytoplasmic domain of Band 3 (cdb3)  contains a dimer interface connected by a domain-swapped β-sheet that extends into a helical segment often referred to as the ‘dimerization arm’16. The monomeric cytoplasmic domain, excluding the VR1, reveals a cytoplasmic N-terminal core domain consisting of the intertwined secondary structure elements CR1 and CR2. The intrinsically disordered region (VR2) forms a connecting linker between CR1 and CR216. A more recent structural model of the cdb3 lacking residues 1 to 55 has been determined at 2.2 Å resolution and crystallized under near physiological conditions (pH 6.5) (PDB ID: 4KY9). Structural comparison with the previous model at pH 4.5 shows only minor molecular differences mainly present on the domain surface17. Transport regulation by the SLC4 HCO3− transporters exerted by the N-terminal domain has been described previously2. However only a few publications, so far, describe that divalent metal ions like Mg2+ and Zn2+ can modulate the transport activity20. The electrogenic activity of NBCe1 was inhibited by Mg2+ (Ki of 0.01 mM) in whole-cell patch-clamp studies18, and the transport activity by AE1 was predicted to be stimulated by intracellular levels of Mg2+ 19. AE2 show partial inhibition by extracellular Zn2+ when expressed in Xenopus oocytes20 while equivalent concentration of extracellular Zn2+ had no effect on AE1 activity20. Here, we present the crystal structure of the truncated  N-terminal cytoplasmic domain from human NDCBE-D (ntcNDCBE) at 2.8 Å resolution. Structural analysis confirms the dimerization interface of previous structural models and determines that the key dimerization motif is a minimal beta-sheet that lacks the residues equivalent to the dimerization arm in cdb3. We identify and describe a novel Zn2+-binding site and the completely conserved HHH motif also found in the electroneutral sodium-bicarbonate transporters (NBCn1 and NCBE) surrounded by a shell of negatively charged residues on the surface of ntcNDCBE. Zn2+-binding properties are verified in vitro for ntcNDCBE and, combined with cellular assays performed on NCBE and NBCn1, this indicates a general Zn2+ dependence that could expand to all members of the SLC4 superfamily.The regulation and maintenance of intra- and extracellular pH homeostasis is fundamental for life. The main buffering system in humans is based on the equilibrium between HCOet al.16, the dimerization of ntcNDCBE is only dependent on the backbone interactions of the four-membered β-sheet and the possible additional hydrophobic interactions between Leu108, Leu110, Phe114, Val337, Leu340 and Val342 from each monomer. The initial dimerization interface proposed for cdb3 included an α-helix – located downstream of β10, residues 327 to 348 in AE1 (PDB ID: 1HYN) – as the dimerization arm, which was claimed to be important for dimerization16. The buried area between the 1HYN subunits is of 2500 Å, while ntcNDCBE has a buried area of 870 Å . However, the molecular interactions of these α-helices are not symmetric, as one would expect for a dimer interface21; we therefore propose, based on the present study, that the dimerization arm only plays a minor role in stabilizing the dimer interface  forms a dimer in the asymmetric unit. Each monomeric ntcNDCBE forms an α-β sandwich fold by nine α-helices and ten β-strands Fig. . The mai22. This arrangement would naturally lead to a high local concentration of single cytoplasmic domains. High local concentrations also occur during crystallization. However, to establish whether ntcNDCBE forms a dimer in solution at lower concentrations, the construct was subjected to size-exclusion chromatography (SEC), and eluted at volumes corresponding to the hydrodynamic volume of a dimer  share an overall similar fold . However, dimers of these two structures superimpose poorly despite the equivalent dimer interface, although they clearly show a pivotal movement between each monomer. By superimposing the central β-sheet  of the individual monomers of cdb3 (chain P) and ntcNDCBE (chain A), a pivotal movement of ~23° is observed between each monomer, while the central dimerization domain-swapped β-sheet  remains intact. This pivotal freedom hints that dynamic flexibility is allowed between the two monomers .The monomeric structures of ntcNDCBE and cdb3  are a specific class of bacterial enzymes that belong to the nitrogen-metabolic phosphotransferase system and have high structural homology with cdb32, particularly E. coli IIANtr . These proteins function as cytosolic regulators of membrane K+ and sugar transporters24. The fold of the cytoplasmic domain in the SLC4 family is unique; however, it does show some structural homology to IIANtr, as described previously2. The lowest RMSD score from the structural alignment was achieved by aligning all the residues of IIANtr with CR2 (α7-9 and β7-9) from ntcNDCBE . Each injection produced an exothermic reaction that decreased in magnitude with subsequent injections. The affinity of ntcNDCBE for Zn2+ was determined to originate from three equivalent binding sites, with an approximate KD of 2.3 µM and experimental thermodynamic parameters ΔH = −6130 ± 110.4 cal/mol and ΔS = 5.24 cal/mol/deg  motif, which has also been found in other Zn2+-binding proteins25, was found surface-exposed on the structure of the cytoplasmic domain of NDCBE  increase in tryptophan fluorescence as a function of Zn2+ concentration  and a double mutant . A strong reduction in tryptophan fluorescence yield was observed for all mutants  are protonated and thus unable to form a complex with any metal ions. Attempts to increase the pH after the crystals had formed or fast soak high concentrations of ZnCBE Fig. . In the CBE Fig. . The HXHCBE Fig. . Sequencily Fig. , whereasily Fig.  and thusion Fig. . To confine Fig. . The sigine Fig. . To exclnce Fig. , the incnts Fig. , suggest167 Fig. , which ives Fig.  show tha2+ on NBC activity on intracellular pH regulation in mammalian cells two setups were investigated. First, the effect of Zn2+ on isolated slc4a10 activity was determined in NIH-3T3 fibroblasts stably transfected with slc4a10. The Na+ and HCO3− dependent NBC activity (dpHi/dt) in the cell line was reduced by 70% in the presence of 10 µM ZnCl2 (n = 5 (control) and n = 4 (ZnCl2), p = 0.0253, unpaired t-test, Fig. slc4a10 activity. Furthermore, the pH recovery in isolated rat mesenteric small arteries  was 0.019 ± 0.002 sec−1 (n = 5) and 0.013 ± 0.001 sec−1 (n = 5), in the absence and presence of Zn2+, respectively .To investigate the physiological effect of ZnSLC4 member is available. The recent crystal structure of the AE1 transmembrane domain only allows speculation with regard to which surface of the cytoplasmic domain dimer face the transmembrane dimer. It is common for most proteins that the core is higher conserved compared to the surface-exposed regions27. The conserved surface residues that form structural patches hint at functional importance. Earlier in this paper, we presented evidence that the dimer interface found in the cdb3 dimer is also present in the here-described ntcNDCBE dimer. The presented structural evidence thus indicates that the dimer interface is indeed physiologically relevant. The dimers all form a two-fold rotational axis that is perpendicular to the β10 A/β10B sheet, leaving only two surfaces in the dimer with conserved residues facing the same direction, as described for typical homodimeric proteins such as superoxide dismutase and interleukin 1028. In ntcNDCBE, one of these surfaces clearly displays patches of higher conservation compared with the other  has a predicted affinity in the low micromolar range37. This affinity is in the same range as the identified Zn2+-binding site of ntcNDCBE, which suggests the possibility of a specific role for the observed interaction between Zn2+ and the N-terminal cytoplasmic domain of NDCBE in presynaptic vesicles. The impact of Zn2+ on NCBE activity suggests that the effect of Zn2+ binding is to reduce NBC activity. This interpretation was supported by the finding of an inhibitory effect of 10 µM Zn2+ on recovery from acidosis in smooth muscle cells of rat mesenteric small arteries most likely mediated by an inhibitory effect on NBCn1.Variations in intracellular Zn2+ binding to the regulatory domain of NBC’s suggests an overall regulatory role of Zn2+ on membrane transport of acid/base equivalents. In the case of NDCBE, the Zn2+ binding may aid in the ion transfer across the presynaptic membrane and thus play a role in neuronal signalling. The inherent pH dependence of this Zn2+ site may also reflect the site of pH sensitivity present in the NBCs. As small concentrational variations in intracellular Zn2+ would affect the equilibrium between the histidine site and free Zn2+ ions, and this would consequentely act as a pH sensor.In conclusion, Zn21, we propose the most likely orientation of this domain in relation to the transmembrane domain. We propose that the interface of the N-terminal cytoplasmic domain that interacts with the transmembrane domain is on the opposite side of the dimer compared with that suggested by Gill et al. for ntcNBCe1-A38. This previous suggestion was based on the localization of three lysine and three leucine residues. Of these six residues from NBCe1-A, only Leu152 and Lys157 (as arginine) are conserved. The N-terminal domain residues responsible for the interaction will most likely not interact with the membrane itself but with residues in the transmembrane domain. In the proposed model of NDCBE  and the complete N-terminal core domain (amino acids 40–425) of human NDCBE (SLC4A8) isoform D (uniprot ID: Q2Y0W8-4) were purchased (GenScript). The Quikchange Lightning Site-Directed Mutagenesis kit (Agilent) was used to mutate the ntcNDCBE constructs used in this study, yielding ntcNDCBE (amino acids 40–344), ntcNDCBE_H167A, ntcNDCBE_H169A, ntcNDCBE_H167A, H169A and ntcNDCBE_W80F. The ntcNCBE and ntcNDCBE constructs were N-terminally tagged with a hexa-histidine tag and TEV protease cleavage site, which were present in the vector. E. coli BL21 Gold (Agilent) was used as the expression strain for ntcNDCBE and ntcNCBE constructs. The cells were grown by fermentation in terrific broth (TB) medium supplemented with 25 µg/mL kanamycin to an OD600 of 2.0 at 37 °C. Protein expression was induced with 0.3 mM IPTG at 18 °C O/N. The cells were harvested by centrifugation at 48,000 × g. All subsequent steps were carried out at 4 °C. Cells were lysed in buffer A  and 1 µg/mL DNase I) using an Emulsiflex high-pressure homogenizer (Avestin). The lysate was cleared by centrifugation at 48,000 × g for 20 min and applied to a HisTrap HP column  pre-equilibrated in buffer B . The protein was eluted with buffer C (buffer B supplemented with 300 mM imidazole) and dialyzed overnight against buffer B in the presence of TEV protease  against buffer B to cleave the his-tag. The cleaved construct was loaded onto an IMAC column pre-equilibrated in buffer B. The flowthrough fractions were concentrated to 10 mg/mL  and subjected to SEC on a Superdex S200 column  equilibrated with buffer D . The peak fractions were pooled and concentrated to 10 mg/mL. The constructs were treated with EDTA  and dialyzed against buffer E  for use in downstream experiments. Purification was evaluated by SDS-PAGE.Codon-optimized pET-M11 plasmids encoding the N-terminal core (amino acids 93–426) of human NCBE (SLC4A10) isoform B (uniprot ID: 40 in XIA241. Molecular replacement (MR) was performed with phaser in CCP442. A truncated version of the cdb3 crystallographic model (PDB ID: 1HYN)16 was used as the MR search model. The model was built using Coot43 and refined in PHENIX44. The final data collection and refinement statistics are presented in Supplementary Table Purified ntcNDCBE was crystallized via sitting-drop vapour diffusion at 4 °C (40 mg/mL) with 0.1 M formic acid, pH of 4.4, and 9% w/v PEG 8000 and was cryo-protected with 20% ethylene glycol. X-ray diffraction data were collected from a single crystal at the BESSY II 14.1 beamline. The data were processed using XDS2 (Fisher Scientific), MgCl2, CaCl2 and EDTA. The measurements were performed in triplicate on a Jasco FP8500 spectrofluorometer at 25 °C with a stirring speed of 200 rpm and a 30 sec delay between injection and measurement. The cell path length was 10 mm. The excitation wavelength was 295 nm, and emission spectra were recorded from 310 nm to 500 nm. Data were corrected for the dilution factor. The increase in tryptophan fluorescence (F) yield was calculated at the wavelength corresponding to the maximum intensity of the fully Zn2+-saturated curve (336 nm). The increase in tryptophan fluorescence yield due to Zn2+ titration was calculated using equation . The titrants used were ZnClbound is F when the construct is fully saturated, and Ffree is F without any metal added. The decrease in tryptophan fluorescence yield due to EDTA titration was calculated using equation  Hill equation, equation :3\documeKD is the dissociation constant.where B is the maximum value of F, h is the Hill coefficient, and 2+ were determined by ITC in a MicroCal ITC200 system  at 25 °C. The sample cell was filled with 200 µL of degassed protein solution at 50 µM. The titration solution of 0.9–1.5 mM metal in dialysis buffer was titrated in 2 µL injections with a 90-sec delay between injections. Background subtraction was performed using the appropriate buffer. Data integration and curve fitting were performed with the Origin ITC analysis package. ITC experiments were repeated at least twice for each construct, with similar results in both the number of sites and the affinity range.Purified, EDTA-treated ntcNDCBE and ntcNCBE were dialyzed against buffer containing 100 mM NaCl, 20 mM BIS-TRIS pH 7.2, and 0.2 mM TCEP. The affinities of the constructs for Zn45. Based on sequence conservation, ntcNDCBE was divided into VR1 (1–39); CR1 (40–128); VR2 (129–236); and CR2 (237–344). The resulting alignment was mapped onto ntcNDCBE and the transmembrane domain of AE1  using the online server ProtSkin46. The residue conservation score was based on sequence identity.Representative isoforms of each human NBC and AE of the SLC4 family were aligned using MUSCLEQ2Y0W8-4; NBCn1 – Q9Y6M7-1; NCBE – Q6U841-1; NBCe1 – Q9Y6R1-1; NBCe2 – Q9BY07-1; AE1 (1HYN/4KY9) – P02730-1; AE2 – P0492-1; AE3 – P48751-1.Uniprot identifiers: NDCBE – Slc4a10-transfected NIH-3T3 fibroblasts were previously described47. The cells were stably transfected with a plasmid containing the full length mouse slc4a10 construct . Cells were cultured in Dulbecco’s modified Eagle’s medium glutaMAXTM supplemented with 10% donor bovine serum. For pHi measurements cells were grown on glass coverslips.4Cl prepulse for 5 minutes followed by a Na+ free bicarbonate buffered salt solution ). The Na+ dependent HCO3− transport was determined as the dpHi/dt after switching to a bicarbonate buffered Na+ containing solution (BBS) containing 10 µM EIPA to inhibit Na+/H+ exchange activity. Each measurement ended in a one-point calibration at pH 7.0 in a high K+-solution containing 10 µM nigericin. In separate experiments, the fluorescence ratio was calibrated to pH by clamping pHi stepwise from 6–7.5 in a high K+ HBS with 10 µM nigericin48.Prior to measurements the cells were loaded with 2 µM BCECF-AM (ThermoFisher scientific) in a HEPES-buffered salt solution for 10 minutes at 37 °C and mounted in a closed perfusion chamber (Warner Instruments). The chamber was perfused with a linear flow rate of ~0.8 mm/s at 37 °C and placed on an inverted microscope stage in a 37 °C dark box. The fluorophore was excited with alternating 495-nm and 440-nm light from a monochromator (Till photonics). The light emission at 510–535 nm was recorded by a 12-bit cooled monochrome CCD camera  and data was collected from regions of interest of a minimum of 3 cells per coverslips after background correction. Cells were allowed to equilibrate to a baseline level in HEPES buffered salt solution (HBS) followed by acidification by a 20 mM NHHBS: 145 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 139 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 HPO42−; 20 mM NH4Cl solution: 20 NH4Cl, 125 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 139 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 PO42−; Na+free BBS: 24 HCO3−, 24 choline, 121 N-Methyl D glucamine, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 115 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 HPO42−; BBS: 24 HCO3− 145 Na+, 3.6 K+, 1.8 Ca2+, 0.8 Mg2+, 115 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 PO42−. All solutions were adjusted to pH 7.4. Bicarbonate buffers were bubbled with 5% CO2. High K+HBS: 10 Na+, 139 K+, 1.8 Ca2+, 0.8 Mg2+, 139 Cl−, 0.8 SO42−, 5.5 glucose, 10 HEPES, 2 HPO42−.The solutions contained (in mM): i of rat mesenteric small arteries was measured as described previously50. Eleven week old rats were killed by CO2 inhalation where after a segment of a rat mesenteric small artery  was dissected free and mounted in a myograph for isometric force recording. The arteries were kept in a solution (PSS) containing (in mM): Na+ 143, 4 K+, HCO3 25, 1.6 Ca2+, 1.0 Mg2+, Cl 130, 2 HPO42− 2.0, 10.0 HEPES, glucose 5.0). The solution was gassed with 5% CO2 in air and the temperature kept at 37 °C. VSMC pHi was measured using wide-field microcopy of arteries loaded with 5 µM BCECF-AM (Invitrogen) for 30 min. The preparation was placed on the stage of a microscope and the fluorophore excited with alternating 435 nm and 488 nm light. The emission at 510–535 nm at each excitation wavelength was recorded by a photometer and the ratio of the emissions corresponding to excitation at 488 and 435 nm determined. Intracellular acidification was induced by adding 15 mM NH4Cl for 15 min followed by washout into a Na+ free PSS (NaCl was substituted with equimolar amounts of N-methyl-D-glucamine titrated with HCl while NaHCO3 was substituted with equimolar amounts of choline bicarbonate). After 5 min Na2+ was washed in (the Na+ free PSS substituted with PSS) and the rate of increase of the emission ratio was taken as a measure of pH recovery. This value was determined under control conditions, in the presence of 10 µM Zn2+ and again after washout of Zn2+. The pH recovery in the presence of Zn2+ was compared to the mean pH recovery in the control condition and after washout of Zn2+.pHSupplementary data


In the field of numerical cognition it is often highlighted that the domain-specific systems, referred to as “Approximate Number System” (ANS), or “The Number Sense” (NS)Performing both simple numerical processing tasks as well as advanced mathematics requires involvement of domain-general processes apart from domain-specific components. The input material has to be perceived and preprocessed, if necessary stored in (working) memory, processed, and a decision needs to be made. Eventually, all this may lead to a response being given. For the importance of such processes see e.g., LeFevre et al.  and RousTo provide an overview, in the following paragraphs we present three examples that cover domain-general factors operating at varying levels of information processing: from visual grouping through strategic attentional allocation up to inhibition. We discuss how these domain-general processes influence varied areas of numerical cognition, starting from dot set comparisons/estimations through multi-digit number processing up to calculation. These examples are not exhaustive but are aimed at showing the tight relationship between domain-general cognitive factors and number processing at different levels.Despite spatial arrangement of digits being irrelevant to the operation order from a content point of view, Landy and Goldstone  found thSumming up, visual grouping processes that are evident in several domain-general cognitive tasks also play a considerable role in number processing at different levels, and to-be-inhibited visual stimuli are semantically processed, influencing task performance.Multi-digit number processing , participants tend to focus on decade magnitude, and the effect of unit-decade compatibility is reduced or disappears totally, because the irrelevant unit causing interference is rarely attended  than the role of domain-specific factors  on “Linguistic Influences on Numerical Cognition: A cross-cultural investigation using natural specificities of Polish and German languages.”The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The objectives of this study were to determine the herd and within herd prevalence of Bovine Viral Diarrhoea Virus The average number of cows tested per herd was 35.5 (median 30). Herd level seroprevalence to Bovine Herpesvirus-1(BHV-1), Bovine Viral-Diarrhoea Virus (BVDV), Leptospirosis and Neosporosis was 90%, 100%, 91% and 67%, respectively, while the mean within herd prevalence for the these pathogens was 40%, 77.7%, 65.7% and 5.7%, respectively. The study confirms that the level of seroconversion for the four pathogens of interest increases with herd size. There was also evidence that exposure to one pathogen may increase the risk of exposure to another pathogen.Herd level seroprevalences were in excess of 90% for BVDV, BHV-1 and Leptosporosis. Larger herds were subject to increased exposure to disease pathogens. This study suggests that exposure to several pathogens may be associated with the further exposure to other pathogens. In recent years, the issue of herd health and, in particular, infectious disease has received increasing prominence as a potential factor influencing reproductive efficiency in Ireland and elsewhere. However, despite its obvious critical importance, there are limited published data available, to quantify the influence of various endemic diseases on beef cow and herd fertility. Reproductive performance in beef cow herds is related to several management, physiological and disease factors , 2. The Leptospira interrogans hardjo-bovis and Neospora caninum are four of the most significant pathogens associated with infertility and abortion in Irish cattle. While non-infectious agents probably contribute more to overall reproductive wastage in cattle, there is limited information on the extent and importance of infectious disease on reproductive performance in Irish beef cow herds.Numerous bacterial, viral and protozoan pathogens have been associated with infertility and abortion in cattle. BVDV, BHV-1, BVDV is a pestivirus which is endemic in the cattle population of several countries and is responsible for a wide range of clinical syndromes, amongst which are immunosuppression and early embryonic death . In receBovine herpes virus 1 (BHV-1) is associated with clinical syndromes such as rhinotracheitis, pustular vulvovaginitis and balanoposthitis, abortion, infertility, conjunctivitis and encephalitis in cattle , 8. It eLeptospirosis is a bacterial disease of cattle that is characterised by reproductive failure in the form of abortions, stillbirths and birth of weak calves. It is also a zoonotic disease. In cattle, the leptospires of most interest are interrogans hardjo-bovis and -prajitno genotypes (i.e. sub-serovars). A number of studies have reported reduced conception rates in seropositive dairy cows , 13.A prNeospora caninum typically occur during the early second trimester, but may occur throughout gestation. A number of authors have suggested that cattle infected with the parasite are two to 12 times more likely to abort compared with uninfected cattle [Abortions due to d cattle , 16. Thed cattle . In a lad cattle . A recend cattle  As far aThe objectives of this study were to (i) determine the herd and within herd prevalence of BVDV, BHV-1, Leptospirosis and Neosporosis in a large cohort of commercial beef herds on the island of Ireland, and (ii) examine herd level factors associated with exposure to these pathogens in these herds.In all, 6049 cows in 161 beef cow herds from across the island of Ireland were sampled in this study. Of these, 156 were commercial beef cow herds, including 25 participating in national extension programmes. The remaining five herds were located on state owned research facilities. Blood sampling took place once for each herd over the summer months of either 2014 or 2015, and only spring calving cows within the herds were sampled. Data on herd vaccination and mineral supplementation policies were gathered at the time of sampling.All experimental procedures involving animals were licensed by the Irish Health Products Regulatory Authority (HPRA). Protocols were conducted in accordance with the Cruelty to Animals Act  and the European Community Directive 86/609/EC and were sanctioned by the Teagasc Animal Ethics Committee. Blood samples were collected via the coccygeal vessel into 10 ml non additive containers  for serological analysis.Sera were centrifuged and aliquots for each test were partitioned into serum vials and frozen at −20 °C pending analysis. Sera were screened using commercially available ELISA kits for BVDV, BHV-1, Leptospirosis and Neosporosis in Department of Agriculture, Food and the Marine Regional Veterinary Laboratory service (RVL). In each case the tests were carried out according to the manufacturer’s instructions.For herds where IBR vaccination was reported, the kit used for BHV-1 was the IDEXX IBR gE kit . The manufacturer’s report a sensitivity of 98.41% and a specificity of 99.76%. The cut off for a positive sample was an SN (sample to negative control ratio) value of <0.6.In herds that did not declare an IBR vaccination policy the testing kit used for BHV-1 was the IDEXX IBR gB X3 . The cut off for a positive sample for this kit was >55% blocking. The manufacturer’s report a sensitivity of 100% and a specificity of 99.8%.The kit used for BVDV antibody was the Svanovir BVD – Ab Bovine Virus Diarrhoea Virus antibody Test . The cut off for a positive result was a PP (percent positivity) value >35. The manufacturer’s report a sensitivity of 100% and a specificity of 98.2%.L.hardjo antibody kit . The manufacturer’s report a sensitivity of 99% and a specificity of 85%. The cut off for a positive sample was a PP (percent positivity) value of >45%.The kit used for Leptospirosis antibody was the PrioCHECK The kit used for Neosporosis antibody was the IDEXX Neospora antibody kit . The manufacturer’s report a sensitivity of 97.56% and a specificity of 98.53%. The cut off for a positive sample was a S/P (sample to positive ratio) value of >0.4.n = 139) was accessed from the Irish Cattle Breeding Federation (ICBF) database for the years 2013, 2014 and 2015. This information was not available for the herds located in Northern Ireland.The herd level BVDV status for each of the Republic of Ireland herds . Details of the herd demographics for Republic of Ireland herds are presented in Table Herd level registration and movement data were obtained from the Irish Department of Agriculture, Food and the Marine’s Animal Identification and Movement (AIM) database and the following variables were calculated for each herd: herd size, number of cows, overall mortality rate, calf mortality up to 30 days of age, calves per cow per year and purchase rate. These variables and herd level prevalence of the various serological tests were calculated and entered into the spreadsheet, in addition to herd vaccination history. Statistical analysis was carried out using Stata 14 . A descriptive statistical analysis of the data was initially conducted. In order to examine the potential for regional differences the herds were divided into seven geographical regions  in Region 1 to 172.0 (median 135.0) in Region 4  Republic of Ireland herds since the inception of the compulsory BVD programme in the Republic of Ireland  for the first time within an Irish context. This is one of the first studies in Europe to integrate data from the national cattle identification and movement database with disease prevalence data, to identify risk factors associated with the occurrence of BHV-1, BVDV, Leptospirosis and Neosporosis.Increasing herd size emerged as a factor associated with seroconversion to BHV-1, BVDV and Leptospirosis, and the disclosure of BVDV positive animals. Herd size has previously been documented as a significant risk factor associated with exposure to these pathogens in Ireland , 14, 20.There were numerous associations identified between the pathogens under investigation. For example, the seroprevalence of BHV-1was associated with the seroprevalence of Neosporosis, and the disclosure of BVDV positive calves in the herd. Indeed, the role of co-infection has been previously described by Van Leeuwen et al. , where cIn addition to herd size and associations with other pathogens increased BHV-1 seroprevalence was associated with increased overall herd mortality rates. Previous studies have reported herd level seroprevalences of 77% and 74% in Northern Ireland and the Republic of Ireland, respectively , 20. ThePrevious studies have described herd level seroconversion to BVDV in excess of 98% in both the Republic of Ireland and Northern Ireland , 20. SimThe disclosure of BVDVpositive calves was related to the seroprevalence of BHV-1and herd size. One third of the Republic of Ireland study herds participating in the national BVD eradication scheme reported one or more BVD virus positive calves in the initial 3 years of the scheme . The disIn addition to herd size, the seroprevalence of Leptospirosis was associated with the level of livestock mart purchases in 2014. It is difficult to determine the significance of this, as it was only found to reach statistical significance in 2014. This association is likely to be biosecurity related, and was associated with movement of animals between holdings and spread within holdings.In this study there was evidence of seroconversion among 90% of non-vaccinating herds, which was greater than the 82% herd level seroprevalence described in a previous Irish study of beef cow herds  Table 77. The meNeospora caninum has previously been associated with the presence of dogs and wild canine species such as foxes on the farm [The herd seroprevalence for Neosporosis of 67% reported here is almost identical to that of a similar study in Australian beef cow herds , which athe farm , 16. In the farm .The farmers that participated in our study volunteered to do so and were not evenly distributed across the island of Ireland. Region did not emerge as a significant variable in any of the multivariate analyses and the disease prevalences in the various regions were broadly similar. While this study may not be representative of beef cow herds overall, participating herds came from a wide range of sizes, which enabled us to adequately investigate the effect of herd size.While there are few perfect serological tests available, the tests that were used in this study were of a high sensitivity and specificity. We are confident that the tests BHV-1, BVDV and Letporosis provided areflection of the sero status of the animals tested. However, given the dynamics of the host parasite interaction and the characteristics of neospora serological tests, it is probable that the Neospora ELISA underestimated the true prevalence of Neosporosis in the cows tested .The level of seroconversion to BHV-1in Northern Ireland may have been over estimated, as conventional non marker BHV-1vaccines are still available on the market there. For this reason, data from herds located in Northern Ireland were not included in the linear model, and the BHV 1seroprevelance in the various regions was calculated individually as we avoided using a cumulative figure so as to prevent any one region biasing the overall results.Seroconversion is a reflection of historic exposure to a particular pathogen, and is not necessarily an accurate reflective of infection status of an individual animal. However, a high herd seroprevalence, especially among younger animals in a herd, may be suggestive of active infection in that herd. This study was herd based and could not take account of the age of the animals within those herds. The issue of individual animal factors will be addressed in a parallel piece of research . Only seropositive animals were included in calculating the herd seroprevalence, and animals with an inconclusive test result were treated as seronegative as this was a herd level based study.Vaccine usage was much higher among herds in this study than in previous studies. This may be a reflection of the targeted nature of participant selection, whereas previous studies selected herds randomly. The farmers in our study volunteered for inclusion and were therefore likely to be more motivated than the general population.Our findings support those of several previous studies that as herds increase in size there is increased exposure to pathogens. Consequently farmers with larger herds need to be aware that they must increase their efforts to avoid the introduction and spread of pathogens to their herds, to establish the health status of their herds and determine what mitigation measures are required to address those diseases.This study has also shown that exposure to several pathogens is associated with exposure to other pathogens. This suggests that there is a common factor common to the exposure among these herds, which is likely to be related to biosecurity. This also indicates that biosecurity needs greater attention in larger herds.


The Ti-based MGMC has excellent tensile properties and pronounced tensile work-hardening capacity, with a yield strength of 1100 MPa and homogeneous elongation of 4%. The distinguished strain hardening is ascribed to the formation of deformation twinning within the dendrites. Twinning generated in the dendrites works as an obstacle for the rapid propagation of shear bands, and then, the localized necking is avoided, which ensures the ductility of such kinds of composites. Besides, a finite-element model (FEM) has been established to explain the TWIP effect which brings out a work-hardening behavior in the present MGMC instead of a localized strain concentration. According to the plasticity theory of traditional crystal materials and some new alloys, TWIP effect is mainly controlled by stacking fault energy (SFE), which has been analyzed intensively in the present MGMC.The present study demonstrates that Ti-based metallic glass matrix composites (MGMCs) with a normal composition of Ti However, they generally fail in a brittle manner under uniaxial quasistatic loading at room temperature2. To alleviate this challenge, a series of in-situ dendrite-reinforced metallic glass matrix composites (MGMCs) with large tensile ductility have been developed, such as Ti-based and Zr-based MGMCs5. However, most of these composites exhibit softening rather than work hardening after yielding upon tension at room temperature, associated with localized necking. In contrast, generally, the traditional crystalline alloys such as steels, Ti as well as Al alloys8, and new promising crystalline alloys such as high-entropy alloys (HEAs)9, have outstanding tensile work-hardening capacity. And the excellent properties are induced by transformation-induced plasticity (TRIP) and twinning-induced plasticity (TWIP) effects, which is closely related to stacking fault energy (SFE)10. Referring to MGMCs, up to now, the work-hardening capability mainly relies on the micro-mechanisms of TRIP effect, including the martensite transformation in CuZr-based MGMCs13, and the transformation from β to α phase in Ti-based MGMCs15. However, for most in-situ MGMCs including thermally stable dendrite-reinforced composites, the work-hardening is rarely available4, unless the volume fraction of stable secondary phases takes value very high so that a network and even disconnected structures of amorphous phases form16. In other words, the irreversible phase transformation favors the availability of work hardening in MGMCs after yielding, which has been the sole way to acquire the flow stress larger than yielding stress so far.Bulk metallic glasses (BMGs) are recognized as potential structural materials due to their superior performance, such as high strength, large elastic limit, and excellent corrosion and wear resistance, etc.17. The present composite with secondary dendrites not only settles the softening shortcoming appearing in the common MGMCs, but also guarantees the high yielding strength. By investigating mechanical responses under tension, the deformation mechanisms in this study are emphasized on the TWIP effect in β dendrites.In this work, a novel Ti-based MGMCs with macroscopic tensile ductility and work-hardening capacity has been developed. The composites consist of the amorphous matrix and β dendrites with a metastable body-centered cubic (bcc) structure as the secondary phases. Upon loading, the shear bands generated within the glass matrix can be arrested by the ductile dendrites, which avoids catastrophic rupture with highly-localized shear banding and results in excellent fracture toughness. It is noted that the work-hardening behavior induced by the deformation twins formed within bcc dendrites is firstly reported here. And, almost no phase transformation happens within dendrites during plastic flows. Usually, twinning frequently occurs in the TWIP steels, which is related to SFE. When the slip is impeded during plastic deformation, a new slip system would be activated by twinning, leading to a strengthening effect. Thus, the deformation twins can remarkably improve the tensile strength43Zr32Ni6Ta5Be14 were produced by arc melting the mixture of pure elements Ti, Zr, Ni, Ta, and Be with purities greater than 99.9% (wt.%) under a Ti-gettered argon atmosphere. Plate-shape samples with 2 mm in thickness and 10 mm in width were obtained by the copper-mould-casting method. Dog-bone-like plate specimens with gauge dimensions of 10 mm (length) × 2 mm (width) × 2 mm (thickness) were well polished and used for tensile tests. The tension was conducted using a strain rate of 5 × 10−4 s−1. The as-cast and fractured samples were observed by scanning-electronic microscope (SEM) to examine the microstructure and fractographs. Crystalline phases were identified by X-ray diffraction (XRD), transmission electron microscopy (TEM), and high resolution TEM (HRTEM) in a JEM-2010 microscope. The specimens for TEM were prepared by mechanical grinding followed by ion milling. A Nano Indenter II tester  with a trihedral Berkovich indenter was used to calculate the Young’s modulus of both the glass matrix and dendrites at room temperature.Master alloys with a normal composition of Ti43Zr32Ni6Ta5Be14 MGMCs. It can be seen that in-situ precipitated dendritic phase is homogenously embedded in the amorphous matrix. The volume fraction of the dendrites takes a value of approximately 54%, and the analysis of EDS gives an average composition of the glass matrix and dendrites, which are characterized as Ti35.7Zr38.3Ta4.0Ni8.0Be14 and Ti50.3Zr37Ta8.6Ni4.1, respectively. The element Be is considered to only exist in the matrix totally4. The spacing of primary dendrites is about 0.5–1 μm, determined from a higher magnification of the SEM image, as displayed in Fig. Figure in-situ dendrite/MGMCs4. Instead, a regular arrangement of lattice patterns prevails, and no ordered structure is available within the glass matrix.To further exactly identify the dual-phase structure, the TEM technique has been employed to analyze the structure of the as-cast composites. A bright field (BF) TEM image at low magnification is displayed in Fig. Figure in-situ dendrite-reinforced MGMCs reported18.XRD has been conducted on the as-cast and deformed samples to clarify the structure of the composites, as displayed in Fig. 4. As can be seen, parallel deformation bands and symmetrical deformation zones are substantially found in dendrites, denoted by the bright arrows and red lines, respectively. Apparently, the deformation bands are identified to be twinning by TEM analysis. The areas, marked with the red rectangle in Fig. Although XRD results characterize most macroscopic situations within samples, more details should be focused on microscopic scale to unveil the deformation mechanisms. Figure 19. Figure 12. Figure 20. Scarcely, twinning is essentially found in nano- and micro-scaled bcc crystalline alloys such as Ta21, W22, Mo23, and V crystals24. Previously, in in-situ MGMCs, the dendrite phase is mainly composed of Zr-Ti-Nb (V). Accordingly, the dislocation slip rather than twinning is the dominating deformation mode. In the current composite, Ta-rich bcc solution may favor the availability of twinning during plastic flows. Ogata et al.25 have proposed that the variation of SFE has its origins from the electronic structures, and closely relates to the charge density change during the stacking fault formation. According to this criterion, Wu et al. obtained that Ta has very high SFE compared with Co, Ni, Cr, Ag, Ti, and Hf26. However, twinning easily generates in the micro-scaled and nano-scaled Ta crystalline samples during plastic deformation27. And the bulk samples are tested in this study, discontinuous network structure in micrometer-sized dendrites including element Ta is predominant, which facilitates twinning happening in deformed dendrites.Figure in-situ MGMCs with a macroscopic scale exhibit softening or hardening after yielding upon tensile loading. Softening is usually induced by the multiplication of shear bands in the glass matrix, while hardening can appear when the TRIP or TWIP effect reacts within the composites. Figure σf) during plastic deformation, corresponding to the hardening behavior, is larger than yielding stress (σy). And, an opposite case occurs with softening behavior. Here, different from the ductilization mechanisms induced by phase transformation and/or typical particles such as Ta within MGMCs28, the main plastic deformation mechanisms in the current composites are ascribed to deformation twins. Twins can be formed through various approaches including plastic deformation at extremely low temperature and/or high strain rate, phase transformation, thermal treatment, and other physical or chemical processes in a large variety of metals and alloys30. Dipankar et al. and Sun et al. have found that TRIP and TWIP effects can occur in the Ti alloys, which contains a bcc phase32. While, nanoscaled mechanical twins are not easily achieved experimentally under usual deformation conditions in MGMCs33. Neither with an unique structure nor through special processing, the deformation twinning ensures high yield strength and outstanding work-hardening capacity in the present composite. It means that the understanding about deformation mechanisms in MGMCs under tension is limited, and the TWIP effect in MGMCs is discussed later.The deformation behavior in MGMCs containing ductile dendrites under tensile loading is attributed to the unique plastic deformation mechanisms. The deformation mechanisms in all kinds of 35, and the strain-hardening capacity is controlled by the mechanical twins. The deformation twins induced by high tensile stress exert influences on mechanical properties by acting as the effective obstacles for dislocation movement according to the Hall-Petch type strengthening mechanisms. The local deformation is hindered, and the necking phenomenon may be avoided36. It is well known that shear bands are initiated firstly in the amorphous matrix, and then, the ductile dendrites are deformed to hold back the generation of multiple shear bands14. This leads to intense interaction of interface between the matrix and dendrites, which causes a remarkable change in the interfacial transition zone35. It is noted that the shear bands generated in the matrix are concentrated at the boundary, caused by the stress concentration at the interface. And the dendrites that are subject to the high stress easily produce a large amount of dislocations to hinder the propagation of shear bands11. Different from the dislocation activity mechanisms, the deformation twinning here determines the deformation mechanisms. Wang et al. has found the formation of deformation twinning in nanocrystalline Ta21. Oh et al. reported the TWIP effect in Ti alloys containing Ta14, which means that Ta is beneficial for the formation of deformation twinning. From Fig. 37. More significantly, the stress concentration is another crucial factor for the generation of deformation twinning. As shown in Fig. 38. And the deformation twinning could stop the propagation of shear bands, and meanwhile prevent the movement of dislocations. Therefore, the current composite exhibits work-hardening behavior under tensile loading. On the other hand, the deformation twins in crystalline dendrites with different orientations can be considered as the sub-boundaries, which results in high strength in the present composite39. The twins with different orientations penetrate dendrites from the boundary to inside, which are followed by the formation of more twins with increasing the stress. The size of the twinning keeps in nano scale, as displayed in Fig. et al.29. Furthermore, the growth of cracks can be effectively blunt by twins, and the deformation twinning among dendrites prevents the formation of individual mature shear bands. Instead, profuse intersectant shear bands prevail, indicative of distinguished plasticity, which is consistent with fractographs shown in Fig. 40. As mentioned above, the stress concentration in the deformed dendrites induces twins. The nucleation and growth of deformation twins among dendrites can absorb much strain energy, which inhabits the multiplication of shear bands in the glass matrix associated with softening. The dendrites and deformation twins could interrupt the propagation of shear bands, which avoids the catastrophic rupture. The conclusion can be drawn that the deformation twins in dendrites, together with multiple shear bands in the glass matrix, ensure the high strength as well as good ductility in such a kind of composites.The studies about the TWIP effect are mainly focused on the TWIP steelsin-situ dendrites influence the stress distribution within the composites, and lead to a softening or hardening behavior under tension. As required, the deformation behaviors of MGMCs are described by the free-volume and elastic-plastic models, which are incorporated into a finite-element code (ABAQUS) as a user material subroutine41. And the concentration of free volumes, which can be used to characterize the shear bands, is denoted as an internal state variable of SDV1 in ABAQUS. The TWIP effect benefits to the work hardening occurrence as shown in experimental results29. And in order to explain the evolution of shear bands, the multilinear elastic-plastic model is established. The present MGMCs with obvious work-hardening capacity is characterized by the TWIP effect. Guided by the work of Jiang et al.42, the 2-D plane-strain finite-element model (FEM) is developed in this work. Within this model, the loading boundary is coupled, and the displacement loading, which is perpendicular to the loading boundary, is applied at the corresponding reference set. The opposite boundary to the loading boundary is applied to the normal constraints, and a vertex belongs to the opposite boundary is applied to full constraints. It should be noted that although the tough particles such as dendrites are randomly dispersed in the real composites, a regular distribution of elliptic particles still reflects the main characteristics of the composites to some extent. Thus, the dendrites are assumed to be elliptic particles. The material parameters of the present MGMCs are listed in Table 41. Figure in-situ dendrites. Figure in-situ ductile dendrites within MGMCs, in which the black one responses to a work hardening behavior induced by deformation twinning and the other one (red one) shows softening without TWIP effect. The black curve in Fig. 43. The maps of strain contours with a tensile strain of 6% are shown in Fig. The ductility of MGMCs is closely related to the stress distribution between the matrix and dendrites upon tension. The 44. The deformation at different stages is schematically illustrated in the inset of Fig. The above detailed deformation mechanisms have been demonstrated by visualized experiments and a simple FEM. Meanwhile, it is of importance to relate with the mathematically theoretical foundation. According to the stress–strain curve shown in Fig. σ′, E, ε, and εy are the elastic stress, Young’s modulus, elastic strain, and yielding strain of the composites, respectively. Both of the dendrites and glass matrix being elastic are suitable for Eq. , the composite starts to be yielding with increasing strain. However, the dendrites and glass matrix yield asynchronously, and the dendrites yield firstly46, which means the elastic-plastic deformation begins at the second stage. Meanwhile, the stress can be expressed as follows47:σ and σd are the tensile stress of the composites and dendrites, respectively. εp is the plastic strain given as 47, cd is the average stress concentration factor of dendrites, which has a value of 148. Consequently, Eq.  for the formation of the stacking fault is expressed as follows51:γ is the SFE, and b is the Burgers vector of the perfect dislocations. And the critical shear twinning stress (τT) is calculated to be as follows52:bp is the Burgers vector of the partial dislocations. Concerning the critical shear stress (τ), a relationship based on the equation of τ = σnm is established. Here, σn and m represent the normal stress and Schmid factor, respectively. The maximum Schmid factor for deformation twins is 0.5. Therefore, the critical normal stress used to produce deformation twins can be calculated as The stress relation between the composites and dendrites is shown in Eq. , and theσd, overcomes the critical normal stress, σT, the deformation twins in dendrites emerge. Combining Eqs ,,σd, ovebp, in Eq.  systems55, which strongly demonstrates that the stress within dendrites has reached a critical stress to form the deformation twinning, and the twins indeed appear in present work.The composite subjected to tensile loading produces a certain quantity of deformation twins, as can be observed in Fig. , in Eq.  with thefrom Eq.  at the sg to Eq. . The SFE47:The strain goes on with further deformation, and the glass matrixes yield, both of which indicate the coming of the plastic-plastic stage. The tensile stress of the composite is estimated to beEq.  can be e56. Normally, the glass matrix (phase) exhibits strain softening, accompanied by the occurrence of multiple shear bands after yielding57. Nevertheless, the density of shear bands is limited, as shown in Fig. 47. The further explanation about the deformation twins is referred to dislocation reactions. The dislocations are difficult to move due to the hamper of deformation twins, and the perfect dislocations dissociate into partial dislocations due to the formation of stacking faults58. The effect of the dislocations forms new stacking faults and subsequently leads to new deformation twins with other orientations64. The formation of deformation twins in different stages contributes to work hardening of the composites. Shear bands produced within the glass matrix will result in a catastrophic fracture but not for dendrites. On the contrary, the high strength of the glass matrix provides enough stress to induce deformation twins in dendrites.The glass matrix yields, and the following changing of the stress in the matrix makes it possible for the initiation of shear bandsIn accordance with the analysis, the deformation mechanism of the present MGMC upon tension consist of three parts, elastic-elastic, elastic–plastic, and plastic–plastic stages, which usually in accord with the deformation stages (I), (II), and (III), respectively. Within the stage (II) and (III), homogeneous elongation (that is work hardening) can be found, and accompanied by fracture without necking. Homogeneous elongation is extremely necessary for engineering materials subjected to tensile loading, on account of inhomogeneous deformation (that is necking) may reduce the service life of the engineering materials. Obviously, it is of importance for structural applications to appear work hardening in service. The TWIP effect that brings about a pronounced plastic deformation within dendrites promotes the work hardening along with the composite yields. And that, the glass matrix with high yield strength makes sure to achieve a high tensile strength of the composite.in-situ metallic glass matrix composite (MGMC) Ti43Zr32Ni6Ta5Be14 (at %) displays an excellent mechanical performance upon tension at room temperature, achieving a high tensile strength of ~1470 MPa and a plastic strain of ~4%. Both tensile deformation micro-mechanisms and work-hardening behavior, which is induced by TWIP effects, are investigated by experimental and theoretical calculations in this work. The deformation twins found in dendrites acting as plasticity carriers are strongly demonstrated. The essential relations at different stages, the elastic–elastic, elastic–plastic, and plastic–plastic stages that usually matchup the elastic, work-hardening and the finally fractured deformation behaviors, are revealed by theoretical calculations. Work-hardening behavior is necessary for the materials as engineering materials, and the work hardening here is resulted from ductile dendrites and associated with homogeneous plastic deformation in the present composite. Moreover, the high yielding strength of the matrix makes contribution to the high strength of the composites. In consequence, the studies in our present work reveal that both deformation twining occurred within the in-situ crystalline dendrites and high yielding strength of the glass matrix ensure excellent mechanical properties of the composite, which provides a guide to design highly ductile MGMCs which exhibits remarkably homogeneous tensile elongation at room temperature.In summary, the