Quantum key distribution (QKD) networks hold promise for sharing secure randomness over multi-partities. Most existing QKD network schemes and demonstrations are based on trusted relays or limited to point-to-point scenario. Here, we propose a flexible and extensible scheme named as open-destination measurement-device-independent QKD network. The scheme enjoys security against untrusted relays and all detector side-channel attacks. Particularly, any users can accomplish key distribution under assistance of others in the network. As an illustration, we show in detail a four-user network where two users establish secure communication and present realistic simulations by taking into account imperfections of both sources and detectors. Quantum key distribution (QKD) ,2,3,4 prOn the one hand, many previous demonstrations of quantum networks heavily rely on the assumption of trusted measurement devices. From security point of view, however, such assumption is challenging in realistic situations, as various kinds of detector side-channel attacks are found due to the imperfections of practical devices ,14,15,16On the other hand, most of the existing QKD networks are limited to point-to-point QKD. When expanded to multi-partite QKD case, the complexity increases, and the efficiency decreases significantly. Recent study shows that multi-partite entanglement can speed up QKD in networks . TherefoC communication users. Remarkably, this feature allows communication users not to be specified before the measurement step, which makes the network flexible and extendable. Furthermore, the MDI feature enables this scheme to be secure against untrusted relays and all detector side-channel attacks. Specially, all users need only trusted state-preparation devices at hand, while the untrusted relay section is made by entangled resources and measurement devices.In this paper, we propose a flexible and extensible protocol named as open-destination MDI-QKD network, by combining the idea of open-destination teleportation  and MDI-N-party quantum network. We are particularly interested in the case where arbitrary two users want to share secure keys. This scenario is denoted as Consider an The Step. 1Preparation: A third party, which may be untrusted, prepares N-partite GHZ stateZ. All users prepare BB84 polarization states, i.e., X. The third party and all users distribute the prepared quantum states to their relays, which may also be untrusted.Step. 2Measurement: The relays perform Bell state measurements (BSMs). When using linear optical setups, only two outcomes related to projections on Step. 3Announcement: All relays announce their successful BSM results among a public classical authenticated channel. The two communication users announce their photons bases, and other users announce their states prepared in the X basis.Step. 4Sifting: The two communication user keep the strings where all the relays get successful BSM results and other users use X bases. Then, they discard the strings where different preparation bases are used. To guarantee their strings to be correctly correlated, one of the two users flip or not flip his/her bit according to the corresponding BSM results and other users’ prepared states (see Step. 5Post-processing: The two communication users estimate the quantum phase error and quantum bit error rate (QBER) in Z and X bases, according to which they further perform error correction and privacy amplification to extract correct and secure keys.N untrusted relays. In a practical scenario, all the users can use weak coherent pulses to reduce experimental cost and apply decoy-state techniques [In this protocol, the multi-partite GHZ state between distant users can also be established through a prior distributed singlets, following the scheme of Bose et al. . In factchniques ,27,28 toWe will show the correctness and security of the open-destination MDI-QKD protocol, i.e., the communication users end up with sharing a common key in an honest run and any eavesdropper can only obtain limited information of the final key. The following analysis applies for the X-basis states, the two communication users can perform flip their bits locally to obtain perfectly correlated sifted keys. We start from rewriting the GHZ state asFor the correctness of the protocol, we show that after successful BSMs and other users announce the Here, X basis, denoted as a string kth bit k-th particle of the GHZ state. Here, We label each user by Z-basis states, or when they both prepare X-basis states with X-basis states but Here, X-basis states  oZ basis, Z basis, X basis, f is the error correction efficiency, and Similar to the open-destination MDI-QKD in Section  of the (scussion ,44,45. Ascussion , the secscussion ,39,40:(1t states .As a conclusion, we proposed a flexible and extensible scheme of the A significant virtue of our scheme is the security against untrustful relays and all detector side-channel attacks. Moreover, the open-destination feature enables any two users to establish MDI-QKD without changing the network structures. In fact, one can establish MDI-QKD among arbitrary users even after the entangled source have been distributed and all the measurements have been completed. Furthermore, following the multi-entanglement swapping scheme, the network can be extended into a large scale by adding shared multi-partite GHZ states.We would like to remark that currently the efficiency was relatively low (seen from


Lysine (Lys) is indispensable nutritionally, and its levels in plants are modulated by both transcriptional and post-transcriptional control during plant ontogeny. Animal glutamate receptor homologs have been detected in plants, which may participate in several plant processes through the Lys catabolic products. Interestingly, a connection between Lys and serotonin metabolism has been established recently in rice. 2-Aminoadipate, a catabolic product of Lys appears to play a critical role between serotonin accumulation and the color of rice endosperm/grain. It has also been shown that expression of some lysine-methylated proteins and genes encoding lysine-methyltransferases (KMTs) are regulated by cadmium even as it is known that Lys biosynthesis and its degradation are modulated by novel mechanisms. Three complex pathways co-exist in plants for serine (Ser) biosynthesis, and the relative preponderance of each pathway in relation to plant development or abiotic stress tolerance are being unfolded slowly. But the phosphorylated pathway of L-Ser biosynthesis (PPSB) appears to play critical roles and is essential in plant metabolism and development. Ser, which participates indirectly in purine and pyrimidine biosynthesis and plays a pivotal role in plant metabolism and signaling. Also, L-Ser has been implicated in plant responses to both biotic and abiotic stresses. A large body of information implicates Lys-rich and serine/arginine-rich (SR) proteins in a very wide array of abiotic stresses. Interestingly, a link exists between Lys-rich K-segment and stress tolerance levels. It is of interest to note that abiotic stresses largely influence the expression patterns of SR proteins and also the alternative splicing (AS) patterns. We have checked if any lncRNAs form a cohort of differentially expressed genes from the publicly available PPSB, sequence read archives of NCBI GenBank. Finally, we discuss the link between Lys and Ser synthesis, catabolism, Lys-proteins, and SR proteins during plant development and their myriad roles in response to abiotic stresses. As amino acids are building blocks of proteins, they participate in the synthesis of hormones in animals and in peptide hormone synthesis in plants . Reduced sensitivity to feedback inhibition in transgenic plants with overexpressed bacterial DHDPS , which helps to form the phospholipids necessary for signal transduction. L-Ser is not only a proteinogenic amino acid, but also it participates in catalytic functions of diverse enzymatic reactions in plants. Importantly, Ser takes an active part in the biosynthesis of many biomolecules such as amino acids, phospholipids, and sphingolipids that are obligatory for cell proliferation . Ser getThis review discusses dehydration proteins known as dehydrins (DHNs), which are members of the LEA protein family group 2 or D-11 . Since tThermus thermophilus; via the intermediate DAP. This pathway adds carbon groups to aspartate to yield Lys  is a novel variant that catalyzes tetrahydrodipicolinate to L,L-DAP and then helps in the synthesis of Lys in lower plants (Physcomitrella patens) as well as higher plants like A. thaliana, soybean, and spinach  enzymes catalyze the two vital steps in Lys biosynthesis  catalyzed by different enzymes. LL-DAP-AT that directly converts THDPA to LL-DAP in A. thaliana (regarded as DAP-AT pathway). Interestingly, LL-DAP-AT operates efficiently in the forward/biosynthetic direction in plants  pathway  and α-amield Lys , but exhield Lys . Both paield Lys . Glutamaield Lys , darknesield Lys . Some of spinach . This inynthesis . It is an plants . DiaminoP to Lys .Glycine max  that contains both LKR/SDH activities  are accumulated in high concentrations, they may be toxic to the plants , hence, urnished .LKR/SDH and aminoadipic semialdehyde dehydrogenase (AASADH) genes involved in saccharopine pathway , amino acids , phospholipids, sphingolipids, and cell proliferation . Cystein pathway  has been pathway . Photore2) and ammonia (NH3) are formed with the release of NADH from NAD+ (2-THF). Methylene THF now combines with the second molecule of glycine to form L-Ser catalyzed by a Ser hydroxymethyltransferase (SHMT) enzyme (Arabidopsis SHMT mutant (shm1-1) was isolated by 2 . Both carbon dioxide (COrom NAD+ . In a co) enzyme . This is) enzyme . The glyted by 2 . One of thaliana , which eSer is also synthesized in leaves through glycerate pathway. It is well-known that 3-phosphoglycerate (3-PG) is synthesized during glycolytic pathway . L-Ser ivia plastidial glycolysis and Calvin cycles acts as a precursor for the synthesis of Ser (+ is reduced to NADH. 3-PHP is then converted to 3-phosphoserine (3-PS) by a transamination reaction catalyzed by 3-phosphoserine aminotransferase  acts as a precursor for auxin biosynthesis  has been found to act as a signaling molecule between male gametophyte and pistil communications  and salt stresses and in restricting pathogen-induced cell death  by SHMT1 wild type plants. They found that reduced SHMT activity led to a decreased stomatal closure in response to ABA and salt stress. Transgenic lines, which showed reduced SHMT activity, were not only more sensitive to salt stress but also to drought stress recovery. In line with this, via participation of the reactions connected with glycerate and PPSB pathways. Thus, a link between carbon and nitrogen metabolism exists, which is regarded as vital in maintaining cellular redox and energy levels under stress conditions.ll death . FurtherPGDH gene isolated from Aphanothece halophytica  lead to elevated salt and cold stress tolerance in A. thaliana  accumulate less proline and raffinose (stress markers) in roots under salt stress than lines overexpressing PGDH3 (OexPGDH3). Moreover, the levels of oxidized glutathione  increased under salt stress in OexPGDH3 as compared to both OexPGDH1 and wild type plants. Ser is utilized in many biosynthetic pathways and thereby contributes to nucleotide synthesis, methylation reactions, and production of the reducing power. Both glutathione and NADPH help in antioxidant defense in plants  scavenges ROS generated during abiotic stress  has been discovered as an ordered protein with phosphorylation sites , indicating a functional linkage between K- and S-segments. While the motif GXGG is highly pliable and interacts with negatively charged phosphoserines with K-segment, motif RRKK appears to be a nuclear localization signal  and also frequent occurrence of one of such subclasses YnSKn in monocots has been noticed by nSKn members were noticed in a drought-tolerant barley (Sorghum bicolor out of six detected (Medicago truncatula) by interacting with AtICE1 (induces CBF expression 1) has been shown to associate with stomatal development, increasing the drought tolerance by decreasing the stomatal density of transgenic A. thaliana  and abiotic stresses (Late embryogenesis abundant proteins (LEAs) are hydrophilic and thermostable in nature. This property helps them to interact not only with biomolecules, but also metal ions . DHNs aron sites . Hence, stration , hydratestration , chaperostration , membranstration , enzyme stration , and in stration . DHNs costration . K-segmestration . More thstration . DHNs hastration . Withoutstration . Howeverstration . While Kstration . On the stration . Stretchn signal . In betw and SKn . While Ystresses . The pret barley , and thrdetected . DHNs hathaliana . DHN5 whstresses , 2016 bestresses .n and YnKn types , desiccation, and salt stresses. The His-flanking K-segments  have been found to bind to membranes and play a major role during stress response  only, which is known to play a regulatory role. Among these ncRNAs, long non-coding RNAs (lncRNAs) are known to be involved in regulation of gene expression. However, very little is known about the role of ncRNAs in providing tolerance against biotic stress including plant diseases. The interactions of lncRNAs with proteins in plants using a system genomic approach is promising and is relevant to ascertain characteristic trait biology relationships. In the recent past, several databases for genes encoding lncRNAs in plants have become available, which include greeNC , Cantataeostasis , flowerieostasis , photomoeostasis , stress eostasis , fertilieostasis , etc. Theostasis  and reguns PPIs; , etc. Soiscussed . In S. bescences . While a bicolor . These r bicolor . Furthercis- and trans-acting elements associated with the activation/suppression of the biosynthetic pathway genes and subsequent events that take place in the downstream remains elucidated. Finally, lncRNAs appear to play a role in these processes by interacting with serine kinase and several novel proteins. Mapping those miRNAs and lncRNAs, their interaction and the key roles they play during the regulation of these processes is certainly the biggest challenge for future research.Both Lys and Ser are key amino acids, involved in plant ontogeny and also connected with abiotic stress tolerance. The biosynthetic pathways are multiple and complex. Among the pathways that exist, the PPSB appears to play a critical role during growth and development. Diverse mechanisms associated with biosynthesis and catabolism of these two important amino acids however, need to be elucidated and also their interaction with other pathways. Attempts were made to enrich the seeds of rice and others with Lys, but still problems exist with regard to seed morphology. These shortcomings need to be overcome in future in order to make high Lys-containing lines available to the public. Further, it is concluded that Lys is catabolized to serve as an alternative respiratory substrate since photosynthetic activity is impaired under abiotic stress conditions. Thus, plants get detoxified by supplementing the much-needed energy during the time of abiotic stress recovery. This vital function of Lys is in addition to its regular participation in protein synthesis and other metabolic activities. Such functions to the amino acids are novel and were not predicted earlier. Many functions of Lys and Ser during growth and development have come into light recently, though the mechanistic explanations are not clear. Further, Lys- and Ser-rich proteins and their induction during abiotic stress reveals the crucial role they play in a wide spectrum of abiotic stresses. Identification of the PBK wrote the first draft with sections on Serine and split by all other authors. RS developed the figures. PS wrote sections on lncRNAs. PBK, PS, and RP proofread the manuscript before all the agreeing to submission. All authors contributed to the revisions and approved the submitted version.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.


It is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. This creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. There have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. Various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. While short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. We review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the International Union of Immunological Societies (IUIS) Veterinary Immunology Committee (VIC) Immune Toolkit Workshop at the 12th International Veterinary Immunology Symposium (IVIS) in Seattle, USA, 16–19 August 2019. The future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for One Health initiatives. This set the scene for a series of VIC Toolkit Workshops  and the Scottish Executive Environment and Rural Affairs Department (SEERAD) in 2003. This was unique as it united several laboratories within a single project to take a collective multi-species approach to immunological reagent development. This was followed by the Veterinary Immune Reagent Network (VIRN) funded by United States Department of Agriculture (USDA)/National Institute of Food and Agriculture (NIFA) in the US in 2005. Both projects included the creation of databases listing available veterinary immunological reagents, which will be discussed later. They also expanded the emphasis from mAb anti-CD antigens to expression of immune proteins (cytokines and chemokines) and protein reactive mAbs. The US project included direct collaboration with commercial partners to express these immune proteins. The US and UK projects worked together under a Memorandum of Understanding (MOU) to avoid duplication of effort. This MOU was created in the absence of a mechanism for joint international funding by the respective national agencies. The structure, priorities and achievements of these projects has been published previously . A key oThe funding for veterinary immunology reagent development has changed over the past 10 years, moving from the multi-species models of the UK Immunological Toolbox and US VIRN, to single-species projects. With the exception of ruminants, there is very little species cross-reactivity of veterinary reagents, highlighting that the genes involved in immune responses are amongst the most rapidly evolving in vertebrate genomes , 23. HowBox 1BMGF: Livestock Antibody Hub: Cattle, swine, poultry (2019–2024):https://www.pirbright.ac.uk/news/2019/11/bill-melinda-gates-foundation-funds-development-pirbright%E2%80%99s-livestock-antibody-hub. To study cattle, pig and poultry antibody responses at high resolution to expand the understanding of protective immunity in those species and that can also be used as models for a range of human infectious diseases.USDA/NIFA: Cattle (2019–2022):https://portal.nifa.usda.gov/web/crisprojectpages/1016686-immune-reagents-for-ruminants-with-primary-focus-on-bovine-specific-reagents.html. To develop, and make commercially available, mAb reagents needed to elucidate cattle immune mechanisms by focusing on CD antigens, cytokines, and chemokines and relevant assays.USDA/NIFA: Swine (2019–2022):https://portal.nifa.usda.gov/web/crisprojectpages/1019192-development-of-new-swine-reagents-to-broaden-our-understanding-of-immune-correlates-of-protection-and-microbial-pathogenesis.html. To generate priority reagents for swine immune proteins and pipeline them for marketing. Develop SLA class I tetramers and new assays for important swine immune markers.USDA/NIFA/BBSRC (US-UK Collaborative): Swine (2015–2019):https://gtr.ukri.org/projects?ref=BB%2FM028232%2F1https://portal.nifa.usda.gov/web/crisprojectpages/1005670-us-uk-collaborative-swine-immune-toolkit-development-of-new-immune-reagents-for-swine-health-vaccine-and-disease-studies.html. To develop panels of mAb reactive with swine targets  using conventional and phage-display methods. Use resultant mAbs to develop new assays for swine immunity and make the reagents commercially available.USDA/NIFA: Horse (2015–2019):https://portal.nifa.usda.gov/web/crisprojectpages/1005524-equine-immune-reagents-development-of-monoclonal-antibodies-to-improve-the-analysis-of-immunity-in-horses.html. To develop and characterize mAbs for the analysis of horse immunity and distribute these to the scientific community for immunological research.USDA/NIFA: Aquaculture (2016–2020):https://portal.nifa.usda.gov/web/crisprojectpages/1009003-collaborative-immune-reagent-network-for-aquacultured-species.html. To develop and provide immunological tools and assays to the aquaculture community to advance health for four fish species: rainbow trout, Atlantic salmon, channel catfish and Nile tilapia.USDA/NIFA: Poultry (2017–2022):https://reeis.usda.gov/web/crisprojectpages/1012306-development-of-poultry-immune-reagents.html. To identify chicken immune molecules, particularly cytokines, chemokines and cell surface markers, express them as recombinant proteins, and characterize their function. Develop mAbs to the target molecules and use these for multiplexed detection assays.BBSRC/SG/BioRad: Cattle and Sheep (2012–2015):https://bbsrc.ukri.org/research/grants-search/AwardDetails/?FundingReference=BB%2FI019863%2F1. To develop reagents and techniques to enable the investigation of the activation and regulation of the immune systems of cattle and sheep with specific reference to cell-surface molecules, intracellular transcription factors and cytokines that can define phenotypically-distinct macrophage, dendritic cell (DC) and T cell subsets.USDA/NIFA: US Veterinary Immune Reagent Network (2010–2015):https://portal.nifa.usda.gov/web/crisprojectpages/0221344-us-veterinary-immune-reagent-network.html. To clone, express, develop mAb reagents specific for ruminants, swine, poultry, equine and aquaculture species, sharing methods across species. Work with commercial partner to market expressed proteins for use by veterinary immunology community.http://tinyurl.com/hxxq3ur)  .The current landscape of the veterinary immunology toolbox has been shaped by new funding approaches to facilitate reagent development while also addressing the complex issues of database construction, collection and validation of data, and sustainability of the database and biobanks of the reagents listed therein. This report summarizes the outcomes of several international workshops where these various elements have been considered.Before summarizing those outcomes, it is worth reviewing the scope of the toolbox in terms of species coverage and knowledge of immunological capability within those species. In the broadest sense, the concept of a veterinary immunological toolbox encompasses a broad range of livestock, companion animal, biomedical model and wildlife species. There has been progress in reagent development across all of those species in the past 10 years which has been presented at various meetings and workshops. We have identified a number of published articles where reagent availability for different species have been reviewed. For the purposes of the toolbox, livestock species can largely be regarded as belonging to one of four major groupings, namely swine , 25, rumhttps://bbsrc.ukri.org/about/reviews/scientific-areas/1506-veterinary-vaccinology-strategy/) and the creation of the BBSRC UK Veterinary Vaccinology Network (VVN).To date, the concept of the veterinary immunological toolbox has largely (but not exclusively) focused on reagent development for livestock species due to their strategic relevance for funders with a stake in livestock health, food safety and global food security. In the period between the last published review of the IUIS VIC Toolkit Workshop at the 9th IVIS in Tokyo  and the http://www.vetvaccnet.ac.uk/publications/veterinary-immunology-toolbox-meeting-uk-veterinary-vaccinology-network). At this workshop, The Pirbright Institute and The Roslin Institute at the University of Edinburgh announced plans for a new UK Immunological Toolbox project. The combined project would be underpinned by core Institute funding from the BBSRC, with additional support from the BBSRC GCRF Tools and Resources . This project is addressing major gaps in capability and sustainability. The first of these is the creation of a publicly accessible, searchable database of veterinary immunological reagents to be accessed via a dedicated website. A follow up meeting was held at the VVN Conference in Stirling in early 2018 (https://www.vetvaccnet.ac.uk/news/2018/01/uk-veterinary-vaccinology-network-conference-2018-report) to discuss in more detail the focus of the website and new reagent development. It was agreed by the community that a key driver for the website would be the facility for researchers to submit information on reagent performance and request reagent production where gaps exist. It was discussed that the primary focus of new reagent development should be around T cell and B cell subsets to help dissect in more detail pathogen and vaccine responses. As well as new reagent development the toolbox aims to exploit new technologies to translate current hybridoma stocks into gene blocks via sequencing and create a recombinant antibody pipeline, express recombinant proteins (including cytokines and chemokines), build multiplex platforms and develop high-throughput screening systems for new antibodies. These sequences act as the template from which the constant region can be switched between different species while maintaining target specificity.In 2017, BBSRC VVN hosted a workshop to discuss the toolbox initiatives in the UK and US with specific relevance to the aims and objectives of the newly-formed Global Challenges Research Fund (GCRF) International Veterinary Vaccinology Network (IVVN). A full report is available on the BBSRC VVN website (https://iuis.org/committees/vic/). The workshop covered the major projects in Europe and the US on reagent development, including a presentation on the plans for the new UK Immunological Toolbox. In the panel discussion, there was broad international support for the approaches being taken within the new toolbox project and recognition of the complementary work being supported by USDA/NIFA in all of the target species (A toolkit workshop was held at the 6th European Veterinary Immunology Workshop (EVIW) conference in Utrecht, Netherlands in 2018. Although this conference was organized under the auspices of the European Veterinary Immunology Group (EVIG), as opposed to IUIS VIC, the IUIS VIC Toolkit Committee took a leading role in the organization of the toolkit workshop. Notably, the toolkit workshop was structured to reflect four newly-formed major livestock groupings  of IUIS VIC Toolkit which were announced for the first time at this meeting. The leaders of the species groups represented their respective areas at the workshop. They are listed on the IUIS VIC webpage and can be contacted by members of the community who are seeking information or looking to engage in reagent development for each of those areas . This database was built around the original information collated during the 2003–2009 BBSRC SEERAD-funded UK Immunological Toolbox and is therefore skewed toward three of the four major livestock groupings . However, aquaculture species, companion animals and now major animal pathogens are also included, and as the community engages the amount of information will increase. The main aim of the website is to collate reagent information and act as a centralized source to increase information exchange but is not the only source for any particular species. For example, the USDA Porcine Translational Research Database (http://tinyurl.com/hxxq3ur) is considered a very wide ranging and valuable community resource and cannot be duplicated but information is shared with the UK Immunological Toolbox via mutual awareness and direct communication.The IUIS VIC Toolkit workshop at the 12th IVIS in Seattle was the forum for the international launch of the Pirbright/Roslin UK Immunological Toolbox website and the associated database  but are generally very adaptable to any given species and synergise well with existing mouse recombinant antibody expression methods. These methods are providing a completely new route to identifying antibodies against specific epitopes on pathogens as well as other foreign immunizing antigens. These antibodies can be used as reagents, including mapping complex epitope landscapes to inform structural vaccinology approaches to increase efficacy, and may also be used as therapeutics. Antibodies are now a primary therapeutic goal of many companies for a range of human diseases. Cats and dogs are not only a profitable target market for immunotherapeutics, they provide value data on function . AlthougThe impact of veterinary immunology research will ultimately be measured by the development, or contribution to the development, of disease-control solutions including diagnostic tests, vaccines and genetic-based strategies. The range of vaccine-delivery platforms is rapidly expanding, including improved adjuvants, vector-based delivery systems and genetic vaccination with DNA and RNA. Although viral-vectored vaccines are successfully deployed in humans and companion animals , public Animal genetics can provide insights into responses to infection and vaccination which can be translated into livestock breeding programmes , 54. BreThe veterinary immunological toolbox is very broad in its scope and has evolved from multiple efforts across the globe. In the broadest sense, the toolbox incorporates livestock, companion animals, wildlife and biomedical animal species. Each is important in its own right, but all are collectively important for the One Health agenda and for controlling existing and emerging diseases that infect different animal populations and have zoonotic potential. As human populations expand, there is a need to protect food security without compromising food safety. Disease prevention and control results in improvements in animal health and welfare, which not only has economic and ethical benefits but can also address concerns for climate change by making food production more efficient. Basic immunology underpins these approaches, from vaccine design to understanding the effects of gene editing. The immunological toolbox website and associated searchable database provides a new focal point for information and knowledge exchange for the veterinary immunology community. The key to future success is global collective working facilitated by networks such as national immunological societies, EVIW, IVVN, American Association of Veterinary Immunologists (AAVI), and IUIS VIC Toolkit Committee.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Shigella sp. vs E. coli and the species of the M. tuberculosis complex). Consequently lipids, along with proteins are also molecules of interest. Lipids are ubiquitous, but their structural diversity delivers complementary information to the conventional protein-based clinical microbiology matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based approaches currently used. Lipid modifications, such as the ones found on lipid A related to polymyxin resistance in Gram-negative pathogens , not only play a role in the detection of microorganisms by routine MALDI-TOF mass spectrometry but can also be used as a read-out of drug susceptibility. In this review, we will demonstrate that in combination with proteins, lipids are a game-changer in both the rapid detection of pathogens and the determination of their drug susceptibility using routine MALDI-TOF mass spectrometry systems.MALDI-TOF mass spectrometry has revolutionized clinical microbiology diagnostics by delivering accurate, fast, and reliable identification of microorganisms. It is conventionally based on the detection of intracellular molecules, mainly ribosomal proteins, for identification at the species-level and/or genus-level. Nevertheless, for some microorganisms  extensive protocols are necessary in order to extract intracellular proteins, and in some cases a protein-based approach cannot provide sufficient evidence to accurately identify the microorganisms within the same genus (e.g.,  In recent years, matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometry (MS) has revolutionized the field of microbiology . MALDI-TIn addition to proteins, lipids are also major cellular constituents. Lipids display high structural diversity and complexity, including species-specific characteristics which enable them to act as useful biomarkers for microbial identification. Although lipids have been used to characterize microorganisms since the 1960s , the iniThis review starts with a short overview of routine MALDI-TOF MS analysis and some of its current constraints, followed by the introduction of species-specific lipids and a brief history of lipid analysis for microbial identification. Afterwards, we will discuss the recent applications of mass-spectrometry based lipidomics for identification of microorganisms and detection of antibiotic resistance. We finish with an outlook toward the future directions for MALDI-TOF MS in microbial lipidomics.In recent years, analysis of intracellular proteins signatures by MALDI-TOF MS has become a routine tool for the characterization of microorganisms . Two comm/z 2,000 to m/z 20,000 in the positive ion mode. This range includes the ribosomal proteins that are present at high abundance in the cell. The mass spectra are then compared to a database that contains the profiles of known microbial species , which is a typical constituent of the cell membrane . LTA is pyogenes . Even thpyogenes . Type IIhosphate . Type IV species .M. tuberculosis, while trehalose polyphleate is present in non-tuberculosis mycobacteria (NTM) species , which are a class of glycosphingolipids uniquely found in plant and fungal species, are particularly important biomarkers due to their high structural diversity . GIPCs apoptosis . The conpoptosis .As well as lipids from Gram-negative bacteria, Gram-positive bacteria, mycobacteria and filamentous fungi lipids, archaebacteria lipids have the potential to provide lipid-specific biomarkers. In contrast to glycerol-ester phospholipids from bacteria and filamentous fungi archaebacterial membranes are composed of isoprenoid and hydroisoprenoid hydrocarbons and isopranyl glycerol-ether lipids . AlthougAs far as we are aware, routine identification of microorganisms by MALDI-TOF MS based on the identity and abundance of lipids has not been explored extensively to differentiate species and subspecies. This represents an exciting area which deserves more investigation.Gaiella occulta  or gas chromatography mass spectrometry (GC/MS) . Both li occulta . As a reEnterobacteriaceae and some Gram-positive species . The MIS is an automated gas chromatographic system that identifies microorganisms based on the analysis of fatty acids that are between 9 and 20 carbons in length. The MIS automatically names and quantitates fatty acids enabling computerized identification of bacteria and fungi by comparing the fatty acid profile of the sample to profiles of known species in the database. It achieved a relatively high accuracy of identification for a range of microorganisms, including mycobacteria and non-fermentative Gram-negative bacteria .Despite the popularity of fatty acid analysis, this method has not reached clinical microbiology laboratories due to several limitations. The major drawback of gas chromatographic analysis of fatty acids is the time-consuming and laborious sample preparation. Before gas chromatography analysis, fatty acids are released by saponification and then methylated to generate fatty acid methyl ester (FAME) derivatives. FAMEs have increased volatility which facilitates their analysis by gas chromatography . This reAnalytical techniques including electrospray ionization (ESI) MS and Direct Analysis in Real Time (DART) MS have also been used as alternatives to GC-MS for fatty acid profiling . HoweverMALDI-TOF MS is a powerful analytical tool with applications in microbiology not limited to protein profiling. Over the last decade many groups have developed MALDI-TOF routines for monitoring the degradation of antibiotics such as carbapenems and indicators of antibiotic resistance . Severalin situ as the lipid extracts are added directly to the MALDI target plate spotted with CeO2. The technique, termed metal oxide laser ionization (MOLI) MS, obtained reproducible species- and strain-specific fatty acid profiles of isolates belonging to Enterobacteriaceae, Acinetobacter and Listeria genera. The accuracy of the method, validated by multivariate statistical methods, reached 100% for species-level classification and 98% for strain-level classification with only one strain being identified incorrectly. Particularly important for clinical microbiology, MOLI MS correctly identified the isolates of Shigella, the bacteria routinely classified by protein profiling as E. coli naphthalene (DMAN), has also been utilized for lipid fingerprinting of intact Gram-positive  strains . The use strains .However, a novel method developed by Larrouy-Maumus et al. enables direct MALDI-TOF MS analysis of lipids on intact microbes . A majorAs antibiotic-resistant bacteria emerge worldwide, the timely detection of antibiotic resistance is critical for appropriate therapeutic management and effective infection control. One of the most promising applications of lipid-based MALDI-TOF MS analysis is the detection of colistin resistance. Colistin is one of the polymyxin antibiotics used as a last-resort therapy for treating multidrug-resistant Gram-negative bacterial infections . Resistam/z) of the major lipid A peaks. The addition of pETN to a phosphate group at position 1 of lipid A results in an m/z +123 shift of the native lipid A-related peak, while L-Ara4N modification causes an m/z +131 shift. This test can also discriminate between chromosome- and MCR-mediated resistance as L-Ara4N modification is generally observed in strains with chromosome-encoded resistance, while pETN modification predominates in strains expressing MCR  and methicillin-susceptible S. aureus (MSSA) strains , Schenk and colleagues reported on the analogous distribution of altered fatty acids and glycerophospholipids, from E. coli strains, upon exposure to sublethal concentrations norfloxacin, an antibiotic that belongs to the class of fluoroquinolone antibiotics .GL-M is a co-inventor of the MALDIxin test for which a patent has been filed by Imperial Innovations. MK is an employee of Bruker, the manufacturer of the MALDI Biotyper system.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Setting land aside has long been a primary approach for protecting biodiversity; however, the efficacy of this approach has been questioned. We examined whether protecting lands positively influences bird species in the U.S., and thus overall biodiversity. We used the North American Breeding Bird Survey and Protected Areas Database of the U.S. to assess effects of protected and multiple-use lands on the prevalence and long-term population trends of imperiled and non-imperiled bird species. We evaluated whether both presence and proportional area of protected and multiple-use lands surrounding survey routes affected prevalence and population trends for imperiled and non-imperiled species. Regarding presence of these lands surrounding these survey routes, our results suggest that imperiled and non-imperiled species are using the combination of protected and multiple-use lands more than undesignated lands. We found no difference between protected and multiple-use lands. Mean population trends were negative for imperiled species in all land categories and did not differ between the land categories. Regarding proportion of protected lands surrounding the survey routes, we found that neither the prevalence nor population trends of imperiled or non-imperiled species was positively associated with any land category. We conclude that, although many species (in both groups) tend to be using these protected and multiple-use lands more frequently than undesignated lands, this protection does not appear to improve population trends. Our results may be influenced by external pressures , the size of protected lands, the high mobility of birds that allows them to use a combination of all land categories, and management strategies that result in similar habitat between protected and multiple-use lands, or our approach to detect limited relationships. Overall, our results suggest that the combination of protected and multiple-use lands is insufficient, alone, to prevent declines in avian biodiversity at a national scale. Reduction in habitat quantity and quality is the major cause of species extinctions and endangerment –4. HenceLands managed to conserve biodiversity in the U.S. have been divided into two broad categories on the basis of the level of protection: (1) protected land—land designated with the explicit intent of protection of native land cover types often with the stated goal of preserving biodiversity, ecosystem services, and cultural values , and (2)Although extensive, protected and multiple-use lands in the U.S. consists of thousands of discrete land parcels amalgamated through piecemeal designations stemming from political, social, and conservation objectives , 11, 18.Past efforts to evaluate the efficacy of the protected and multiple-use lands in conserving biodiversity examined the extent to which these lands represent species  or A more direct and informative approach to evaluate the efficacy of protected and multiple-use lands is to compare species population trends and prevalence among protected, multiple-use, and undesignated lands  at presence of protected and multiple-use lands ; and (2) there was an effect of proportion of area of protected and multiple-use lands . We hypothesized that imperiled species prevalence should be higher on, and population trends should be positively correlated with, protected lands more than multiple-use or undesignated lands. These hypotheses are grounded on the explicit but untested assumption that protected land is preserving avian biodiversity better than multiple-use or undesignated lands.Populations of many bird species in the U.S. have declined and those declines are often attributed to the loss, degradation, and fragmentation of habitat –36. We wWe used data from the USGS BBS and the USGS Gap Analysis Project (USGS-GAP) Protected Areas Database of the U.S. (PAD-US) to compare long-term population trends and species prevalence among protected, multiple-use, and undesignated lands in the U.S. All spatial data layers were projected to USA Contiguous Albers Equal Area Conic USGS (North American 1983 datum). We used the PAD-US  to diffTo evaluate the efficacy of protected and multiple-use lands, we used count data from the BBS across the contiguous U.S. (CONUS). The BBS is a long-term (> 50 years), continent-wide monitoring program that provides standardized survey data for breeding bird populations on >4,000 roadside survey routes. Most BBS routes are surveyed annually, and observers count the number of birds detected for each species at 50 sampling points at 0.8-km intervals along a 39-km survey route .We delineated a 2000-m radius buffer  around each BBS survey route that met our criteria for assessing prevalence and population trend  were nearly identical to their proportions across CONUS (7% for protected land and 19% for multiple use land) based on PAD-US . This inWe defined imperiled species as those whose populations are declining enough to warrant recognition by national or international organizations. Hence, we identified imperiled species as those that appeared on one or more of the following three lists: U.S. Fish and Wildlife Service Birds of Conservation Concern ; Internath meridian) to assess whether our results differed regionally. This split allowed us to examine whether there were regional differences in the efficacy of protected and multiple-use lands while still meeting our criteria for minimum species frequency (see below). Splitting the data further into smaller ecological or conservation regions, as in Wood et al. [Because protected and multiple-use lands are not evenly distributed across CONUS, we created western and eastern subsets  and calculated prevalence as the number of years an individual species was detected on a route divided by the number of years the route was surveyed . Effectively, prevalence, as we have estimated it here, is occupancy without adjustment for detection. We calculated prevalence only for species that were detected on ≥10 BBS routes. We chose a minimum of 10 routes as a conservative estimate of detection of a species on a route. For example, in 10 routes, there is 97% chance of observing a species, even with a 5% chance of detecting it at any stop.To assess the efficacy of protected and multiple-use lands, we examined two metrics related to species persistence: prevalence and population trend . We defiWe estimated route-specific population trends for each species , 48. HowDuring 1966–2014, the amount  of protected land has increased in the U.S. . HoweverFor all spatial analyses, we used ArcGIS 10.x  and for all statistical analyses we used R . An alpha of 0.05 was used for evaluation of statistical significance for all tests unless otherwise noted. Individual tests are described below in their respective sections.To address our first objective, we tested whether species prevalence or population trend differed among routes embedded primarily in protected, multiple-use, and undesignated land for imperiled and non-imperiled species . That isWe estimated mean prevalence and mean population trend for each species across years within each of three categories of BBS routes: (1) ≥50% of the buffer within protected land, (2) ≥50% of the buffer within multiple-use land, or (3) ≥50% of the buffer within undesignated land. We only included species that had routes representing each of these three categories. We calculated the medians and standard errors of the prevalence and population means, respectively. We used Friedman’s test with post-hoc analyses  to evaluWe used three methodological approaches to examine whether the proportional area of protected or multiple-use land affected either species prevalence or population trend among the BBS route buffers  that was protected and multiple-use lands. We also included median elevation, total years the route was surveyed, longitude and latitude at the buffer centroid, proportion of developed land , and proThe analysis of prevalence used the same model.We limited our regression analysis to include only bird species for which we had sufficient data. To do this, we only included species in the regression analysis for which ≥25% of the BBS routes where the species was detected had buffers consisting of ≥5% of either protected or multiple-use lands. Thus, we required that a species occur with a minimum frequency on routes whose buffers contained a minimum proportional area of protected or multiple-use land.We assessed the overall influence of the predictor variables of interest  on the response variables  by comparing multiple linear regression models using Akaike Information Criterion AIC; . Unlike We used McNemar’s chi-square test to determine if the percent of bird species with prevalence and population trend positively associated with the proportional area of protected land within the buffer was greater than the percent of species with prevalence and population trend positively associated with the proportional area of multiple-use land . We examWe used a two-tailed chi-square test to determine which land category was more positively associated with prevalence and population trend. Here, we examined the regression coefficients, associated with proportional area of protected and multiple-use lands within route buffers, to determine which land category was more positively associated with prevalence and population trend . We did As a result of the criteria applied to each dataset, and as an artifact of the differences between the datasets, the total species per analysis group was reduced . Sub-setn = 61, χ2(2) = 11.4, DF = 2, p = 0.003) and non-imperiled  = 28.0, DF = 2, p < 0.001) species. Both species groups were more prevalent on BBS routes embedded in protected  and multiple-use  lands than on routes embedded in undesignated land  or non-imperiled species , regardless of whether the BBS route was embedded primarily in protected, multiple-use, or undesignated lands . We founRegression models assessed the strength and direction of the relationship between proportional area of protected and multiple-use lands and both prevalence and population trend. The relationships between the proportional area of protected and multiple-use lands and both prevalence and population trend varied among bird species  Tables.The proportional area of protected and multiple-use land within route buffers helped to explain variation in prevalence for 38% of imperiled species and 31% of non-imperiled species . Similar2 = 0.03, p = 0.855) or non-imperiled species  or non-imperiled species  or non-imperiled  species, indicating that the effect of protected land is the same as that of multiple-use land regarding species prevalence. See For the analysis of prevalence, only 53% of imperiled species and 49% of non-imperiled species had regression coefficients for protected land that were more positive than regression coefficients for multiple-use land. We found no difference in percent of species in either imperiled . However, we did detect a statistical difference in non-imperiled species ; that is, more non-imperiled species were positively associated with multiple-use land compared to those associated with protected land. Fifty-one percent of imperiled species and 43% of non-imperiled species had protected land regression coefficients that were more positive than the regression coefficients for multiple-use land. See For the population trend analysis, we found no difference in imperiled species  assumption that both of these land categories help prevent extinction and maintains avian biodiversity. However, our hypothesis that species prevalence should be higher and population trend should be positively correlated with protected land more than multiple-use or undesignated lands was not well supported. We found that imperiled species have negative long-term population trends, even on routes primarily embedded in protected and multiple-use lands . Additio2 vs. 9,000 km2), a longer time period (49 years vs 14 years), a larger and more diverse group of species (222 vs. 21) and included a wider suite of vegetative communities compared to Barnes et al. [n = 20 for imperiled species; We detected a difference between protected and undesignated lands for species prevalence for both imperiled and non-imperiled species. This difference may be attributed to several factors: (1) sample size of species or BBS routes, (2) total area of protected land that intersected the routes, (3) types of land-use in undesignated lands, and/or (4) the permanence of land cover types on protected lands , 54. In s et al. . For exaThe persistent negative population trends for imperiled species regardless of the land category surrounding the BBS routes is concerning. Populations of imperiled species appear to be faring no better on protected lands than they are on undesignated lands. Thus, our results suggest that protected lands cannot guarantee protection from population declines or extinction , 57. HowProtected and multiple-use lands benefited many species , as evidenced from the percent of species with prevalence and population trends that were positively associated with the proportional area of both land categories . These rFirst, external pressures in the landscape matrix surrounding protected lands may compromise their integrity and function, thus diminishing their conservation benefit , 58, 59.In our analysis, only eight (~0.3%) routes had buffers completely composed of protected land . These rHaliaeetus leucocephalus) require ~2,160 ha during the breeding season and >1 million ha during the non-breeding season, and red-cockaded woodpeckers  live in family groups that require ~80 ha each [Second, size of a protected lands may affect their capacity to offer suitable home range area as well as buffer the effect of external pressures , 61, 64. ha each –67. Many ha each , 69. FurCentronyx henslowii; [Third, highly mobile species may use a combination of protected, multiple-use, and undesignated lands during their annual cycle and thus may be exposed to a multitude of pressures  affecting population trend , 71. Somnslowii; ).Fourth, management strategies between protected lands and multiple-use lands, although different, may result in similar habitat quality on some lands. Not all lands under multiple-use management are used concurrently, to the same extent, or for the same purposes. In national forests, 9% (~73 million acres) are categorized as ‘reserve forests’ and are not managed for timber harvest, likewise, national forests also contain lands with ‘wilderness’ status ), similaFifth, some factors intrinsic to our dataset may have limited the identification of ecological responses. The imperiled species group, by its composition, may be hiding complexity that is obscuring patterns: (1) it may be difficult to detect differences in species trends when all trends are negative; and (2) many imperiled species are associated with grassland and aridland habitat, yet most of the eastern imperiled species are scrubland species, and thus, there may be complexity covered up by our grouping because the scrubland species would respond well to multiple-use management. Additionally, our approach minimized the likelihood of spurious relationships see , but thoWith the current population declines observed in many bird species, there is an increased importance in protected and multiple-use lands preventing extinction and maintaining avian diversity. We were able to show that both land categories do have a role in sustaining imperiled and non-imperiled bird species over the long term and across landscapes. Future research on the differences in habitat quality between protected and multiple-use lands would provide a better understanding of the role each plays in the maintaining bird populations across their annual cycle. Furthermore, the popularity of citizen science along with continued BBS surveys can provide invaluable data for future assessments of population trends and prevalence of birds within protected and multiple-use lands , 79.Conservation efforts continue to focus on protected lands as the primary tool to protect biodiversity , 7, 80. Because undesignated lands make up 74% of the landscape in the contiguous US , these lS1 Figth. Brackets over bars show pairewise comparisons . Asterisks indicate significant differences between pairs based on Friedman’s chi-square test with post-hoc analysis. Significance was evaluated with p ≤ 0.10 for Friedman’s chi-square tests and p ≤ 0.03 with Bonferroni adjustment for post-hoc analysis. Error bars respresent ± SE. Specific results can be found in Median prevalence for imperiled species and non-imperiled species for Breeding Bird Survey (BBS) routes with ≥50% of protected, multiple-use, or undesignated land within a 2000-meter radius buffer surrounding routes. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S2 Figth. Brackets over bars show pairewise comparisons . Asterisks indicate significant differences between pairs based on Friedman’s chi-square test with post-hoc analysis. Significance was evaluated with p ≤ 0.10 for Friedman’s chi-square tests and p ≤ 0.03 with Bonferroni adjustment for post-hoc analysis. Error bars respresent ± SE. Specific results can be found in Median prevalence for imperiled species and non-imperiled species for Breeding Bird Survey (BBS) routes with ≥50% of protected, multiple-use, or undesignated land within a 2000-meter radius buffer surrounding routes. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S3 Figth.‘Predictor variables included’ indicates models which included proportional area of protected and multiple-use lands as predictor variables in the linear regression models to explain prevalence. ‘Competing models’ were those with delta AIC values <2, and models in which including or excluding the predictor variables did not change the delta AIC value. ‘Predictor variables excluded’ indicates models that did not include the proportional area variables. Additional covariates included proportion of developed areas, proportion of agricultural areas, median elevation of BBS route, total number of years BBS route was surveyed, and longitude and latitude at centroid of buffer. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S4 Fig‘Predictor variables included’ indicates models which included proportional area of protected and multiple-use lands as predictor variables in the linear regression models to explain population trends. ‘Competing models’ were those with delta AIC values <2, and models in which including or excluding the predictor variables did not change the delta AIC value. ‘Predictor variables excluded’ indicates models that did not include the proportional area variables. Additional covariates included proportion of developed areas, proportion of agricultural areas, median elevation of BBS route, total number of years BBS route was surveyed, and longitude and latitude at centroid of buffer. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East .(TIF)Click here for additional data file.S5 Figth. Asterisks indicate significant differences between pairs based on McNemar’s chi-square. Significance was evaluated with p ≤ 0.10. Specific results can be found in Species prevalence may be positively associated with both land categories. Horizontal lines at 50% indicate the null model . Breeding Bird Survey (BBS) routes were buffered using a 2000-meter radius. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S6 Figth. Asterisks indicate significant differences between pairs based on McNemar’s chi-square. Significance was evaluated with p ≤ 0.10. Specific results can be found in Species population trends may be positively associated with both land categories. Horizontal lines at 50% indicate the null model . Breeding Bird Survey (BBS) routes were buffered using a 2000-meter radius. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S7 Figth. Significance was evaluated with P ≤ 0.10 using a two-tailed Chi-squared test. Specific results can be found in Bird Survey (BBS) routes were buffered using a 2000-meter radius. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S8 Figth. Significance was evaluated with P ≤ 0.10 using a two-tailed Chi-squared test. Specific results can be found in Bird Survey (BBS) routes were buffered using a 2000-meter radius. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data  and short-term data ; and by spatial subsets: CONUS , West , and East . West and East subsets were divided by the 98(TIF)Click here for additional data file.S1 TableThe 2000-meter radius buffers were categorized as containing 100%, 75%, or 50% of protected and multiple-use land.(DOCX)Click here for additional data file.S2 TableProportional area was calculated within 2000-meter radius buffer surrounding Breeding Bird Survey (BBS) routes. A plus sign (+) indicates that prevalence or population trend for a bird species was positively associated with the proportional area of protected or multiple-use land within the buffer. A negative sign (–) indicates that prevalence or population trend for a bird species was negatively associated with the proportional area of protected or multiple-use land within the buffer. Duplicate names indicate multiple forms, as designated by the BBS. NA indicates that the species was not used in the analysis as a result of criteria restrictions. Species are listed in alphabetical order by common name.(DOCX)Click here for additional data file.S3 TableProportional area was calculated within 2000-meter radius buffer surrounding Breeding Bird Survey (BBS) routes. A plus sign (+) indicates that prevalence or population trend for a bird species was positively associated with the proportional area of protected or multiple-use land within the buffer. A negative sign (–) indicates that prevalence or population trend for a bird species was negatively associated with the proportional area of protected or multiple-use land within the buffer. Duplicate names indicate multiple forms, as designated by the BBS. NA indicates that the species was not used in the analysis as a result of criteria restrictions. Species are listed in alphabetical order by common name.(DOCX)Click here for additional data file.S4 TableMedian prevalence and population trends for imperiled species and non-imperiled species for Breeding Bird Survey (BBS) routes with ≥50% of protected, multiple-use, or undesignated land within a 2000-meter radius buffer surrounding routes. Data are presented by species group, (Imperiled and Non-imperiled), by temporal subsets (long-term data (1966–2014) and short-term data (1993–2014)), by spatial subsets . Asterisks indicate significant differences between pairs based on Friedman’s chi-square test with post-hoc analysis. Significance was evaluated with p ≤ 0.10 for Friedman’s chi-square tests and p ≤ 0.03 with Bonferroni adjustment for post-hoc analysis. See corresponding graphs in (DOCX)Click here for additional data file.S5 TableColumns, such as +Protected, +Multiple-use, show the number of species that were positively associated with proportion of these lands. See corresponding graphs in (DOCX)Click here for additional data file.S6 Tableth. Significance was evaluated with P ≤ 0.10 using a two-tailed chi-squared test. See Bird Survey (BBS) routes were buffered using a 2000-meter radius. Differences were evaluated with a null hypothesis that the underlying probability of success is 0.5. Data are presented by species group: Imperiled and Non-imperiled; by temporal subsets: long-term data (1966–2014) and short-term data (1993–2014); and by spatial subsets: CONUS, West, and East. West and East subsets were divided by the 98(DOCX)Click here for additional data file.


Out-of-hospital cardiac arrest (OHCA) is a growing worldwide public health concern. Previously, Japan experienced poorer outcomes among OHCA patients than in other high-income countries. In the early 1990s, through policy changes, the Japanese government introduced a task-shift model in pre-hospital care. Some medical practices previously provided by physicians exclusively were delegated to non-physicians, including laypeople. Additionally, we initiated a nationwide data collection system for evaluation. We started a nationwide registry of OHCA patients, a paramedic system to provide advanced life-support care, and basic life-support training for laypeople. In the 2000s, the procedures paramedics could provide were expanded, laypeople were allowed to use automated external defibrillators, and the Utstein style was introduced to the national registry.Consequently, pre-hospital advanced care and bystander first-aid increased, registry-based research contributed to evidence-based practices, and―most importantly―outcomes of OHCA patients considerably improved. These Japanese experiences demonstrate that streamlining pre-hospital care, including bystander interventions and standardized data collection, can improve OHCA patient outcomes. Despite this progress, however, there still exist many issues to be addressed in response to the changing and increasing care demands within Japan’s aging population. Prompt response by bystanders and emergency medical services (EMS) can save a considerable number of OHCA patients; however, such prompt response requires increased awareness and training among laypeople, as well as streamlining the EMS system (3).Out-of-hospital cardiac arrest (OHCA) is a growing global public health concern (5). To tackle this issue, the Japanese government began various measures in the early 1990s based on policy change. The government introduced a task-shift model in pre-hospital care, including advanced life-support procedures, performed by paramedic-level EMS personnel, and defibrillation performed by laypeople using an automated external defibrillator (AED) (7). Previously, these procedures were provided exclusively by physicians. Concurrently, the government started nationwide data collection on OHCA patients for evaluation and research.Japanese OHCA patients experienced worse outcomes than those in other high-income countries Consequently, patient outcomes improved, and evidence from Japan significantly contributed to developing and updating international guidelines. In this paper, we describe policy changes in Japan, which could benefit other countries with low OHCA survival rates due to insufficient pre-hospital care.(8). The Fire Services Act, amended in 1963, mandated municipal fire departments to provide EMS . The Fire and Disaster Management Agency (FDMA) became the national lead agency for EMS and determines policy, guidelines, and training curriculum for pre-hospital care. The policies achieved nationwide EMS coverage as an emergency transport system. Each municipal EMS system has a command center, and the phone number 1-1-9 is used for EMS calls nationwide. Generally, an EMS unit comprises three basic-level emergency medical technicians (EMTs-basic), who have completed 135 hours of training.The emergency care and EMS systems in Japan were organized in the 1960s and 1970s, based on legal and policy frameworks, in response to a rapid increase in road traffic injuries (9) to designate emergency care hospitals where EMS could transport patients. In 1977, MHW (10) created a masterplan to reorganize emergency care hospitals into three levels―primary, secondary, and tertiary―based on their ability to streamline referral networks and enhance hospital capabilities through subsidies. Tertiary emergency care was provided in Emergency Critical Care Centres, accredited by MHW.The Ministry of Health and Welfare (MHW) issued a ministerial ordinance in 1964 Despite vigorous efforts to develop an EMS system and improve hospital care quality, pre-hospital care―including bystander first-aid―was quite primitive in the 1980s. One reason was EMTs-basic could perform only basic life-support procedures, such as bag–valve–mask ventilation and chest compression. At the time, the Medical Practitioners Act did not allow non-physicians to perform medical procedures, including diagnoses and treatments. Even auscultation with a stethoscope was not allowed. Additionally, laypeople were not well-trained in first-aid procedures. Bystanders could do nothing while waiting for an ambulance, and EMTs-basic could do little in the ambulance.(5). These terms imply that those who achieved the return of spontaneous circulation (ROSC) before hospital arrival were not focused on by physicians or researchers (11). In fact, most reports on OHCA patients during this period, were hospital-based and focused on DOA/CPAOA; (5) few studies reported population-based data on OHCA patients (12). Contrastingly, contemporaneous studies in other countries did focus on OHCA patients (5).In the 1980s and early 1990s, Japan’s resuscitation science was still in its infancy. There was no standardized data collection mechanism to provide a basis for evaluation and evidence-based practices. Technical terms were not clearly defined, and various inclusion criteria were used without clear definitions in Japanese OHCA studies. Most physicians and researchers referred to OHCA patients as “dead-on-arrival” (DOA) or cardiopulmonary arrest on arrival (CPAOA) (5). Patients who achieve ROSC before hospital arrival usually have better prognoses; however, they were excluded from DOA/CPAOA studies. However, even taking this bias into account, reported prognoses were poor compared to other countries (5).During this period, studies in Japan reported extremely poor prognoses among DOA/CPAOA, though various endpoints were used without clear definitions due to the lack of standardized data format. Full recoveries, a commonly used endpoint defined as returning to normal social activities, were usually around 1% of treated patients (14). The campaign illustrated the effectiveness of the task-shift model for paramedics in the United States and compared it to the Japanese system (14). However, this required new laws, as the Medical Practitioners Act strictly prohibited task shifting for medical care.Poor outcomes for OHCA patients, combined with a growing number of OHCA patients due to the rapid aging of Japan’s population, increased the need for improved pre-hospital care. An aggressive media campaign, started in 1989 to arouse public opinions, called for a task-shift model in pre-hospital care. This model would delegate some medical practices, such as defibrillation, to EMS personnel  (13), (1(17). Both groups, after comparing pre-hospital care procedures and patient prognoses in Japan with those in other high-income countries, independently delivered the following three recommendations independently in 1990: 1) introducing and strengthening a physician-staffed ambulance system so that physicians could start advanced care at the scene; 2) expanding pre-hospital care procedures provided by EMS ; 3) first-aid training for the general public.To address pre-hospital care improvement, MHW established the Commission for Emergency Medical Care in 1989. FDMA established a workshop group for pre-hospital care in 1990  (8), (13(14). MHW also proposed a new qualification scheme, so that sufficiently trained personnel could provide advanced care . Contrastingly, though recognizing the potential of a physician-staffed ambulance system, the FDMA emphasized the feasibility of the task-shift model, given the shortage of emergency physicians, particularly in rural areas, and the benefits of modifying the existing nationwide EMS system. FDMA proposed expansion of the list of procedures EMS personnel could perform, reflecting positive opinions from the Japanese Association for Acute Medicine and Tokyo Fire Department, the largest municipal fire department.However, MHW and FDMA had somewhat different views regarding the first two recommendations. MHW emphasized physician-staffed ambulances’ potential, reflecting cautiousness toward the task-shift model expressed by both the Japan Medical Association and the Japanese Society of Anesthesiologists. They had concerns regarding the quality of advanced procedures provided by non-physician EMS personnel with limited training  (18). Also, FDMA amended its pre-hospital care guidelines and procedures to introduce EMTs-intermediate, who complete 250 hours of training and can perform more procedures than EMTs-basic  Act was enacted in August 1991, to introduce Japanese paramedics, called ELSTs Ts-basic .(6). Although defibrillation by ELSTs shortens the time to defibrillation, leading to better recovery (20), the benefits of pre-hospital defibrillation were fewer in Japan than other countries in the 1990s, as physician instructions were required (22). Cardiac rhythm conversion―from shockable to non-shockable―sometimes occurred while ELSTs awaited instructions (22).At this time, ELSTs can perform a limited set of advanced life-support procedures on OHCA patients, including defibrillation using a semiautomatic defibrillator, under specific physician instructions via radio communication  (6). Alt(23). Then, MHW amended the Ordinance for Enforcement of the ELST Act so that ELSTs could perform more advanced procedures.Later, in the 2000s, MHW and FDMA established the Joint Commission for ELST services to review the performance of ELSTs and discuss enhancement of their roles . The com(24). This action did not require legal amendments because anyone can perform basic life-support procedures. Municipal fire departments have taken charge of training laypeople.To educate the public on first-aid and cardiopulmonary resuscitation (CPR) skills, FDMA issued training curriculum guidelines in 1993 for laypeople and instructors (25).Another important policy change in the 2000s was AED use by laypeople. Although AED use had been considered a medical practice exclusively provided by physicians, MHW changed its interpretation of the Medical Practitioners Act and stated that one-time AED use in an emergency is not a medical practice if no physician is available. Therefore, it can be performed by laypeople. Thus, layperson use of AED has been permitted since July 2004 The Utstein style, an international data collection standard, was introduced to Japan in the early 1990s. It has facilitated considerable research and policy evaluation on OHCA patients, making international, regional, and before-after comparisons easier. Furthermore, it has expanded researchers’ and clinicians’ viewpoints on treatment for DOA/CPAOA patients in the emergency room to the occurrence, prevention, and pre-hospital care of OHCA patients in communities; thus, the term DOA/CPAOA has become obsolete.(27).FDMA started national registration of all OHCA patients in July 1994 to evaluate emergency care improvements, particularly ELSTs’ performance. The registry has played a pivotal role in describing the nationwide occurrence and prognoses of OHCA patients. However, the registry database did not follow the Utstein style and lacked information on initial cardiac rhythms, etiology, and neurological functions required for standardized international comparisons. Therefore, the Commission for Sophistication of Pre-hospital Care in FDMA recommended the introduction of Utstein style for the national registry in 2003, and FDMA began using the Utstein style in the national registry in 2005 (28). The Resuscitation Council of Asia was established in 2005, representing Asia as a member of the International Liaison Committee on Resuscitation .Introduction of the Guidelines 2000, the first guidelines developed through international collaboration, led Japanese resuscitation experts to recognize the necessity of international cooperation in developing evidence-based guidelines. Subsequently, the Japanese Resuscitation Council (JRC) was established in 2002 as an organization to coordinate domestic activities―such as publication and training―and participate in international activities (18). Candidates must pass the national MHW board examination to be certified as an ELST. To qualify for the board examination, a candidate should either be an EMT-intermediate with at least five years’ experience or 2,000 ambulance service hours and has completed six months of training or has graduated as an ELST candidate from a vocational school or university. ELSTs and EMS units with ELSTs have been increasing ((26).The Japanese paramedic system, ELSTs, started in April 1992  (6), . Particularly, the increasing number of AEDs in densely crowded places, such as train stations, may have resulted in increased probability of bystander AED use for OHCA in Japan (30). Increased bystander defibrillation using a public-access AED on OHCA patients has contributed to improved patient outcomes (32).After the approval of layperson AED use in July 2004, public-access AED deployment and use increased rapidly nationwide  (26), (2(35). Subsequent establishment of large-scale registries  accelerated research that provided supporting evidence to international guidelines. For example, chest compression-only CPR has been shown to be as effective as conventional CPR, with respect to long-term neurological outcomes, based on regional registries (37) and the nationwide registry (39).Research has progressed since the introduction of Utstein-style standardized data collection methods. To our knowledge, the first studies using the Utstein style in Japan were reported in 1993 Prognoses of OHCA patients have gradually improved over the past several decades, as a result of policy changes and subsequent efforts . These i(40). A Good Samaritan law, which currently does not exist in Japan, would dismiss concerns that one may be penalized for failing to save a life. Although, in practice, rescuers acting in good faith will not be penalized (41), such concerns may discourage people from providing CPR. Furthermore, psychological counseling for rescuers is a pressing need, as they often suffer post-CPR stress reactions (43).Despite these achievements, there are still challenges to be addressed in the future . First, (26). Location mismatch may exist, as OHCAs may be occurring in places with no or poor access to AEDs (44). Therefore, we need to identify potential places where OHCAs tend to occur, and determine the actual use of currently deployed public-access AEDs, to minimize the possibility of location mismatch. The current national registry system does not record bystander public-access AED use for cases with non-shockable rhythms. Such cases should also be registered to know total AED use.Second, public-access AED use is still relatively rare, compared to the considerable number of deployed AEDs in public places (45), probably reflecting different pre-hospital care, in-hospital intensive care abilities, and data collection procedures. For example, a few areas with physician-staffed ambulance systems have shown good performance (33). This system, despite its potential, is not feasible to most municipalities, due to insufficient numbers of emergency physicians, as the policy debate indicated. Furthermore, different criteria to determine whether to resuscitate and transfer patients to the hospital  might have contributed to regional variations. The proportion of non-transported or non-resuscitated cases (obvious death) differed considerably by region (35). Therefore, it is crucial to standardize inclusion criteria in the national registry for more accurate evaluation, in addition to equalizing care abilities, to minimize regional variations.Third, there are regional variations in patients’ outcomes (46).Fourth, vigorous evaluation studies of in-hospital intensive care, including extracorporeal cardiopulmonary resuscitation and targeted temperature management, are needed. Such intensive care might improve post-resuscitation outcomes. Currently, however, evaluations are mostly based on observational studies, given the difficulties of experimental studies such as traditional randomized controlled trials, in critical situations. Thus, alternative study designs that may help overcome current difficulties should be considered Finally, redesigned end-of-life care management is needed in Japan’s super-aging society, where most OHCA patients are aged 80 and over, such that resuscitation attempts are performed based on the patient’s wishes. Some patients receive resuscitation attempts at the end of their lives against their wishes, due to insufficient preparation of advance end-of-care directives, insufficient communication between family physicians and specialists in emergency care, and inappropriate ambulance use. In Japan, EMS crews are not allowed to withhold or withdraw resuscitation attempts. Once they receive an EMS call, they are supposed to resuscitate and transport OHCA patients to the hospital, regardless of the patient’s wishes.Outcomes for OHCA patients have improved during the past several decades, reflecting policy commitment to introduce a task-shift model in pre-hospital care. Some of the authority to provide medical care that was previously strictly restricted to physicians was delegated to paramedic-level EMS personnel and even laypeople (AED use). Since some stakeholders expressed caution toward advanced procedures provided by non-physicians with limited training, this process had to progress step-by-step: law amendments, expanding training, evaluation, and re-expansion of delegated procedures. Concomitant nationwide standardized data collection greatly contributed to evaluation. Furthermore, the nationwide database expanded clinicians’ and researchers’ perspectives from hospitals to communities and facilitated research and contribution to evidence-based practices. These Japanese experiences indicate that streamlining pre-hospital care and standardized data collection are crucial to improving OHCA patients’ outcomes. However, despite progress, many issues still need to be addressed to respond to Japanese society changes, particularly the aging population and their increasing health care needs.NoneThis work was supported by a Grant for Comprehensive Research on Life-Style Related Diseases including Cardiovascular Diseases and Diabetes Mellitus from the Ministry of Health, Labour and Welfare, Japan [Grant number: H29-Junkankitou-Ippan-009]. The funding organization played no role in the study; the views expressed in this paper do not reflect the Ministry’s views.We thank Prof. Seizan TANABE for his help in obtaining literature.SN and TS conceived the idea, all authors contributed to collecting information and interpretation of the information, SN drafted the manuscript, and all authors contributed to revising the manuscript.Parts of this study were presented at the annual meeting of the Japanese Society of Reanimatology in 2017  and the annual meeting of the Japanese Association for Acute Medicine in 2018 .


Long-lived individuals are central in studies of healthy longevity. However, few pro-longevity factors have been identified, presumably because of “phenocopies”, i.e. individuals that live long by chance. Familial longevity cases may include less phenocopies than sporadic cases and provide better insights into longevity mechanisms. Here we examined whether long-lived female siblings have a better ability to avoid diseases at ages 65+ (proxy for “robustness”) and/or survive to extreme ages (proxy for “resilience”) compared to sporadic long-livers. A total of 1,156 long-lived female siblings were selected from three nationwide Danish studies and age-matched with sporadic long-lived female controls. Outcomes included cumulative incidence of common health disorders from age 65 and overall survival. Long-lived female siblings had lower risks of some but not all health conditions, most significantly, depression , and less significantly hypertensive  and cerebrovascular  diseases. They also had consistently better survival to extreme ages  compared to sporadic long-livers. After adjustment for the diseases, the association with mortality changed only marginally suggesting central role of better physiological resilience in familial longevity. Due to their consistently better resilience, familial longevity cases could be more informative than sporadic cases for studying mechanisms of healthy longevity. Long-lived individuals are central in studies of the determinants of a long and healthy life. Both genetic and environmental factors contribute to longevity, and research is increasingly more focused on the genetic part –4. As hiPhysical robustness could be defined as the ability to resist a deviation from the normal physical state and avoid an adverse health event . In thisThe long-lived individuals may live longer than the general population for various reasons. They may be more robust than the general population because they are able to avoid major diseases, or they may be more resilient because they are able to better survive after disease onset, or both. They may also live longer simply ‘by chance’. This study investigates whether long-lived female siblings are more robust and/or resilient than sporadic  long-lived women of the same age, and, specifically, whether they have a better ability to (i) avoid the common diseases of the elderly at ages 65+ (proxy for robustness), and/or (ii) survive to extreme ages (proxy for resilience). We compared the cumulative incidence of the 20 most prevalent chronic conditions from age 65 between long-lived female siblings and sporadic long-lived Danish women. We also evaluated the difference in overall survival at the oldest-old ages between the familial and the sporadic long-livers, and examined factors contributing to this difference. Due to a scarce number of long-lived males, we used only data on long-lived females from three studies enrolling Danish long-lived siblings.st, 2006 was 91.3 years (range: 68.8-105.0), with a median age of 91.7 for siblings and 90.9 for sporadic long-lived women (p=0.013). These women were mainly widowed (79.4% in siblings and 76.7% in controls (null Charlson Comorbidity Index (CCI) score, and the numbers were 63.2% and 60.1% for siblings and controls (p=0.282), respectively. The prevalence of the 20 chronic conditions is shown in The study population was comprised of 2,312 long-lived women: 1,156 female siblings and 1,156 controls. The median age on January 1controls ). NearlyThe majority of the women reported medication prescription within the previous month - as median number of prescribed drugs . The proportion of siblings and controls receiving prescriptions was similar ; however, on average, siblings received a lower number of distinct drugs than controls (p=0.047). The prescribed drugs were mainly related to the nervous systems  and cardiovascular .In the unadjusted models, long-lived siblings had a lower risk of hypertensive disease , chronic low respiratory diseases (CLRD) , and depression  than sporadic long-livers . They alAt the end of the follow-up, 1,763 (76.3%) women, 833 (72.1%) siblings, and 930 (80.4%) controls were deceased. Overall survival for siblings and controls was 89% and 83% at 1 year, 66% and 56% at 3 years, and 47% and 36% at 5 years (p<0.001), respectively .Long-lived female siblings had better 5-year overall survival than sporadic long-lived women (hazard ratio (HR)=0.71 95%CI=0.63-0.81) . After aPeople may live long for various reasons including better robustness or better resilience, or both. They may also live longer lives simply by chance. In this study, we examined whether the long-lived female siblings are more robust and/or more resilient than sporadic long-lived women. Our results indicate that the long-lived siblings may be more robust to some health conditions , and less robust to some other (cancer); though only the risk of depression  remained statistically significant after adjustment for multiple testing . Our resFindings from earlier US studies that used samples of LLFS data suggest that long-lived siblings could be more robust to some diseases. Ash et al.  found loIn our study, we found a marginally significant higher risk of cancer among the long-lived female siblings compared to sporadic long-livers. Ukraintseva et al. earlier suggested potential biological mechanisms of trade-offs between extreme longevity and cancer risk, including antagonistic pleiotropic role of some genetic and non-genetic factors in aging and cancer development, which could be applicable to these results . Other sRegarding survival to extreme ages, we observed a better overall survival of female long-lived siblings compared to sporadic long-livers. Similar findings were reported in a Dutch study that also showed better survival of the long-lived siblings at high ages, as compared to long-lived sporadic controls . The assIt should be emphasized that none of the above studies considered indicators of physiological robustness and resilience in the same population, whereas in our large population-based study, we investigated both disease risks and survival to extreme ages in the same individuals. Our results suggest that better physiological robustness (manifested by lower disease risks) does not entirely explain better resilience  of female members of the long-lived families, as compared to sporadic long-livers. And adjustment for the above diseases, to which the long-lived female siblings were more robust, did not change the association, or did so only marginally.We showed that longevity clustered in families can lead to a significantly lower mortality risk at extreme ages compared to sporadic long-livers. This suggests that resilience has similar biological mechanisms in the long-lived siblings influenced by common genetic and other familial factors. Indeed, in the first part of their lives, siblings share many environmental factors . In addition, studies have highlighted the influence of genetic effects on lifespan at older ages, indicating that genetic factors could play a major role in the familial longevity . GeneticThere are some limitations to this study. First, long-lived female siblings were, on average, slightly but significantly older than the sporadic long-lived women even after matching on age. However, our analyses were adjusted for age. This adjustment did not lead to different results in the survival analyses. In fact, the adjustment for age only changed the HR slightly, and the association became stronger. Second, the condition or disease for which the medication was prescribed (indication code) was not available . Not conThe main strength of this work lies in the study design consisting of long-lived female siblings matched on age with female controls from a representative sample of the Danish population. Thus, the registry-based study design permitted avoidance of selection bias. Long-lived siblings and controls were all identified in high-quality National Danish registers, also leading to a large sample size. Finally, the definition of chronic conditions was based on both hospital and medication data . In thisIn conclusion, long-lived female siblings demonstrated better robustness to some health conditions , while increased vulnerability to some other diseases (cancer). Physiological resilience  was consistently better in the long-lived female siblings than in their age-peers from general population. This indicates that the ability to overcome deleterious life events may be more important for extreme longevity than a good health alone, and that avoiding depression is major factor of maintaining physical robustness in familial longevity. A consistently better survival of the long-lived siblings also suggests that resilience may have stronger genetic component in familial longevity, warranting further investigation. Overall, results of this study indicate that long-lived siblings are excellent candidates for healthy longevity studies, and that familial longevity cases could be more informative than sporadic cases for studying mechanisms of longevity. Since health phenotypes such as discharge diagnoses of chronic conditions and medication could not explain better resilience of the long-lived female siblings, a next step can be to focus on other factors that might explain their better resilience, such as response to acute health events, dynamic changes in functional status, cognitive functioning, or psychological factors 11; 20–22]. Cognitive and physical functioning are known to be highly predictive of survival in the very old individuals [–22. CognThe identification of long-lived siblings was undertaken in three nationwide, consecutive studies in Denmark, for which recruitment ran sequentially during the years 2004 to 2009: the Danish Oldest Siblings (DOS) pilot study, the Genetics of Healthy Ageing (GeHA) study , and thest, 2006 were included. Each female sibling was matched with one female control from the Danish population alive on January 1st, 2006 and randomly selected on age (+/- 2 years).Long-lived female siblings enrolled in these studies and alive on January 1The information used in this study was mainly extracted from the Danish national population-based registers presented below.The CRS, which covers the entire population alive and residing in Denmark since April 2, 1968, contains information on each resident’s vital status, sex, place and date of birth as well as familial links  , 26. AllThe Danish National Patient Register (NPR) is a health register established in 1977 , 28. TheThe Danish Cancer Registry (DCR) contains records of all incidences of malignant neoplasms in the Danish population from 1943 onwards . The regThe Danish National Prescription Registry (DNPR) provides individual-level information on dispensed prescriptions for each person resident in Denmark since 1995 , 32. DNPcancers, hypertensive disease, atrial fibrillation and flutter, ischemic heart disease , cerebrovascular diseases (including stroke), diabetes, disturbance in lipoprotein circulation and other lipids, depression, dementia, chronic low respiratory diseases (CLRD), chronic obstructive respiratory disease (COPD), asthma, respiratory allergy, choroid and retina disorders, diseases of eye lens (cataract), glaucoma, hearing loss, ulcers, osteoporosis, arthrosis. Except for cancers, all diseases were identified through the NPR and/or the DNPR using the register-based definitions defined by Hvidberg et al [The first outcome was the cumulative incidence from age 65 of the 20 most prevalent chronic conditions in Danes aged 75 and older : cancersrg et al  , whichever came first. All-cause mortality was defined as death from any cause. Patients still alive were censored at the date of last follow-up.The second outcome was overall survival. Survival time was calculated from January 1st, 2006 from the CRS. The CCI was constructed from hospital data in the 10 years prior to January 1st, 2006. It was based on primary and secondary disease diagnoses recorded in the NPR. In order to capture medical habits and general health in addition to CCI, medication prescription (yes versus no), the number of prescribed drugs and treated organs within the month prior to January 1st, 2006 were considered. The treated organs referred to level 1 Atomic Therapeutic Chemical groups concerned by medication prescription. Prescribed drugs were assessed to evaluate polypharmacy and as a proxy for general health in addition to CCI.Analyses were adjusted for the following factors, usually associated with overall survival in older adults: marital status, Charlson’s comorbidity index (CCI ), medicaThe cumulative incidence between female siblings and controls was compared by performing conditional logistic regression models based on matching data.st, 2006 was described with Kaplan-Meier curves for female siblings and controls separately and compared using the logrank test. Survival analyses were performed using stratified Cox proportional hazards models based on the matching data with and without potential confounding covariates. Proportional-hazards assumption was tested using Schoenfeld residuals.Survival time from January 1The study has been approved by The Regional Scientific Ethical Committees for Southern Denmark (S-VF-20030227) and The Danish Data Protection Agency (# J.nr. 2008-41-1753).Supplementary Table 1