The 2018 Cnidarian Model Systems Meeting (Cnidofest) was held September 6–9th at the University of Florida Whitney Laboratory for Marine Bioscience in St. Augustine, FL. Cnidofest 2018, which built upon the momentum of Hydroidfest 2016, brought together research communities working on a broad spectrum of cnidarian organisms from North America and around the world. Meeting talks covered diverse aspects of cnidarian biology, with sessions focused on genomics, development, neurobiology, immunology, symbiosis, ecology, and evolution. In addition to interesting biology, Cnidofest also emphasized the advancement of modern research techniques. Invited technology speakers showcased the power of microfluidics and single-cell transcriptomics and demonstrated their application in cnidarian models. In this report, we provide an overview of the exciting research that was presented at the meeting and discuss opportunities for future research. Cnidaria  is a phylum of aquatic animals, unified by the presence of specialized stinging cells called cnidocytes . These bhttp://www.cnidarianmodelmeeting.org) was organized to foster the expansion of the cnidarian research community and emphasize the application of modern molecular tools to both classic and emerging cnidarian models. As a successor to the Hydroidfest 2016 meeting [Hydra and Nematostella, appeared in approximately 60% of the abstracts. However, the overall meeting agenda was diverse, with 11 cnidarian species featured during 44 oral presentation sessions and a total of 22 species represented by the 85 submitted abstracts. Nearly all major cnidarian clades were represented  were given by students and postdocs. The environment provided at Cnidofest enabled students and postdocs to showcase their work and interact directly with established researchers in this small, yet vibrant community. With these fantastic young scientists, new species, new technologies, and new ideas, the cnidarian research community is growing quickly and the future is bright. In this report, we provide an overview of the exciting research presented at Cnidofest 2018.Aiptasia, which is colonized by dinoflagellates from the family Symbiodiniaceae [Aiptasia community, her vision of the future of symbiosis research, and her concern over global warming and the accelerating rate of coral extinction.Virginia Weis (Oregon State University) is an inspiring figure in the cnidarian research community. For more than two decades, she has pioneered and promoted coral symbiosis studies using iniaceae , as a moAiptasia larvae. Based on these discoveries, Weis and her collaborators are currently testing symbionts with chemically modified glycans as an attempt to increase their colonizing abilities, with the ultimate goal of rescuing bleached coral populations. Meanwhile, by incubating Aiptasia with high concentrations of thrombospondin type 1 repeat (TSR) peptides, a key component of the complement pathway, Weis and colleagues turned normal symbionts into ‘super colonizers’. These approaches, though preliminary, may help to alleviate challenges in coral conservation. Finally, Weis laid out her vision for a community-wide effort to develop gene manipulation techniques such as CRISPR/Cas9 in both Aiptasia and their dinoflagellate symbionts. Thanks to the efforts of Weis, many well-established biologists such as John Pringle (Stanford) and Thomas Gilmore (Boston University) have adopted Aiptasia as a research organism in their own labs. Meanwhile, a younger generation of Aiptasia biologists are emerging with talent and dedication, highlighted by the exciting oral presentations and posters during this meeting. Emphasizing the need for multiple communities to unite around the complex issue of saving coral reefs, Weis stressed that “it takes a village to save corals…and we must take action before it’s too late.”Weis emphasized the importance of translating findings in basic research into valuable tools for conservation biology. The lectin/glycan interaction and the complement pathway were identified as key players mediating host–symbiont recognition; disruption of these pathways blocks symbiont colonization in Hydractinia species, H. echinata, and H. symbiolongicarpus. These colonial hydrozoans have long attracted attention from cnidarian researchers due to their regenerative and allorecognition capabilities [Hydra and Hydractinia genome portals , which continue to benefit the cnidarian research community. Wai Yee Wong  discussed the high percentage (> 50%) of transposable elements (TEs) found in the Hydra genome; the biological consequence of these TEs is unclear. Wong refined the TE annotations for Hydra using a novel analytic pipeline, RepeatCraft, and then performed comparative analyses across four Brown Hydra species and one Green Hydra species to ask how the expansion of TEs increased the genome sizes of Brown Hydra compared to Green Hydra [Hydra species as compared to the Green Hydra. Wong hypothesized that this lineage-specific LINE family TE expansion could partially explain the drastic genome size increase in Brown Hydra. Joseph Ryan  discussed his efforts to refine phylogenetic relationships within Cnidaria by first constructing a “backbone” phylogenetic tree from 110 species followed by surveying the 18S rRNA sequences of thousands of cnidarian species to add as “leaves” on the tree. This large phylogenetic dataset includes many new taxa and promises to shed light on the positioning of some ambiguous lineages. Ryan proposed to use machine learning to extract biological traits from the literature as a method to further refine the cnidarian tree of life in a trait-based manner. Finally, Steven Sanders  discussed his successful application of CRISPR/Cas9 genome-editing techniques in Hydractinia. As a proof-of-principle for gene knock-in, Sanders used homology-directed repair to insert GFP into the genome at the elongation factor 1 alpha (Ef1a) locus to create a transgenic line with stable expression of an EF1a–GFP fusion protein  presented genome and transcriptome assemblies of two bilities . Schnitzen Hydra , 24. Won results , he was Hydra, which functions in the re-establishment of head organizer activity and restricting tentacle patterning during head regeneration [Hydra with the Notch inhibitor DAPT causes defects in head regeneration, budding, and nematocyte differentiation. An RNA-seq time course during DAPT recovery was used to identify putative direct targets of Notch. Bert Hobmayer (University of Innsbruck) discussed dissecting the function of secreted Frizzled-related proteins (sFRPs) during cnidarian development. To determine if sFRPs antagonize Wnt signaling in cnidarians, similar to their role in bilaterians, Hobmayer tested the expression pattern and function of NvsFRP in Nematostella. He found that NvsFRP is expressed in the aboral apical tuft region of the planula larva in a region that lacks Wnt expression. Furthermore, loss of aboral NvsFRP function disrupts apical tuft formation. Interestingly, NvsFRP can rescue axis duplication in Xenopus when Wnt is overactivated. However, the results of TOP-Flash assays, which are a read-out for β-catenin activity, do not indicate that NvsFRP acts as a Wnt inhibitor. Future work will aim to resolve this discrepancy and help clarify the role of secreted Frizzled in cnidarians. Hongyan Sun  continued the discussion of Wnt signaling by sharing her comparative functional analysis of Axin between Strongylocentrotus purpuratus (purple sea urchin) and Nematostella. The canonical function of Axin is in the β-catenin destruction complex, where it directly binds to β-catenin and promotes its degradation. While the S. purpuratus Axin homolog has a β-catenin binding domain, the Nematostella Axin homolog does not. However, injection of NvAxin into both S. purpuratus and Nematostella embryos disrupts β-catenin nuclear localization. Thus, Sun suggests that additional scaffolding proteins must be involved.Despite their distinctive morphology, cnidarians share many key developmental and disease-related genes with bilaterian animals. In two consecutive sessions centered around cnidarian developmental biology, eight speakers showcased the power of using cutting-edge techniques to address the function of evolutionarily conserved genes and signaling pathways. Angelika Böttger (Ludwig-Maximilians-University Munich) discussed the role of Notch signaling in neration . Böttger6-Methyladenosine (6-mA) modification in Hydractinia. 6-mA DNA modification is a highly conserved feature from ctenophores to mammals, yet its biological function during animal development remains elusive [Nematostella. Vasa-positive PGCs form two clusters, one on each side of the pharynx of a primary polyp. These cells are localized right at the junction between Hedgehog1 and Patched expression domains. Loss of function analysis for Hedgehog pathway members revealed a previously unappreciated role for this pathway in the specification and migration of Nematostella PGCs. Naga Nakanishi (University of Arkansas) performed CRISPR/Cas9-mediated mutagenesis of the GLW-amide peptide in Nematostella. Although these mutants did not display developmental defects, a significant delay in the timing of larval metamorphosis was observed, indicating that GLW-amide exerts control over life cycle transitions in Nematostella [Hydra. Cazet performed ATAC-seq experiments in regenerating head and foot tissue 3 h after injury. Cazet found that an identical generic wound response is activated in both tissues, which takes place largely in the absence of chromatin remodeling in regulatory regions. Thus, he proposed that in Hydra the injury response involves the activation of constitutively open regulatory regions. Finally, Ahmet Karabulut  described his protocol for rapid delivery of shRNA into Nematostella eggs using electroporation [Several talks highlighted gene function in relation to specific aspects of development, including genome activation, the formation of primordial germ cells (PGCs), metamorphosis, and regeneration. Febrimarsa  discussed DNA N elusive –29. Febrtostella . Jack Caporation , 32. ThiRapid developments in genomic and transcriptomic technologies in recent years have enabled a deeper understanding of the genetic composition of cnidarians and of their gene-regulatory landscape. Single-cell RNA-seq (scRNA-seq), for instance, allows identification of rare cell types and transient cell states that occur during development or as stem cells differentiate. These approaches are widely applicable and provide an unparalleled opportunity for cnidarian researchers to uncover biological phenomena otherwise invisible using bulk tissue or mixed cell populations. Our technology speaker, Jeffrey Farrell , shared his expertise in single-cell transcriptomics by discussing his work examining zebrafish embryonic development. scRNA-seq allows for the measurement of single-cell transcriptional status while separating cells from their native spatial context. To address this issue, he introduced Seurat (v1.1), a computational method originally designed to retrieve spatial information of individual cells that he developed in collaboration with Rahul Satija (New York University) . FarrellHydra. The adult polyp undergoes continuous cell turnover, thus providing a platform to study stem cell differentiation and tissue maintenance. By sequencing the transcriptomes of approximately 25,000 single cells, Siebert, in collaboration with Farrell, reconstructed the differentiation trajectories from the three lineages that make up the adult Hydra. The data suggest that neurons and gland cells share a common progenitor cell type in Hydra, a hypothesis that will be the subject of future studies [Nematostella. The Marlow lab pioneered single-cell analysis in Nematostella, recently reporting the completion of a single-cell atlas covering both adult and larval stages [Nematostella, and lay the groundwork for comparative studies with bilaterian animals.Farrells’s talk demonstrated the power of scRNA-seq in a conventional model organism and it sparked spirited discussion. We then heard talks from two speakers who have successfully applied single-cell transcriptomics to cnidarian organisms. Stefan Siebert  discussed scRNA-seq data collected from  studies . Finallyl stages . PlessieHydra lines expressing GCaMP protein in different neuronal subtypes and performed calcium imaging. Hym-121 and Hym-355 positive neurons exhibited different excitation patterns. Noro hypothesized that Hym-121 positive neurons might function as mechanosensory neurons, as they responded strongly to mechanical stress applied using tweezers. Dylan Faltine-Gonzalez  investigated the mechanisms governing patterning of the Nematostella nervous system during embryogenesis. Previous studies have shown that the Notch and MEK signaling pathways play instrumental roles in patterning the Nematostella nervous system [NvAth-like, a key regulator of neural differentiation. He then tested the expression levels of candidate downstream genes to identify potential targets that might play a role in specification of neuronal subpopulations. To test the function of these genes, Faltine-Gonzalez proposed to knock genes down in transgenic reporter animals to visualize the function of each gene in neuronal specification. Jamie Havrilak  focused on the plasticity of Nematostella neurons during regeneration and starvation. Taking advantage of transgenic lines that express GFP in NvLWamide-positive and SerumAmyloid-like-positive neurons, Havrilak observed a positive correlation between the number of neurons along each mesentery and animal body size , has constructed customized microfluidic devices for small organisms to interrogate the connections between neural circuitry and behavior. According to Robinson, the idea of applying microfluidics first stemmed from the frustration over trying to perform electrophysiology in Hydra nervous system activity and behavior. Adult Hydra polyps are comparable in size to adult C. elegans, while possessing a simpler tissue architecture, making them an attractive system for neurobiology research. Krishna Badhiwala  discussed how she and Robinson pioneered real-time behavior tracking in Hydra using customized microfluidics devices [Hydra also exhibited periodicity in locomotion activity, reminiscent of the sleep-like state previously observed in C. elegans.Inspired by his success with worms, Robinson then applied a similar strategy to study  devices . BadhiwaHydra development. These cadherins interact with each other and are crucial for the establishment of planar cell polarity (PCP) in bilaterian systems [Hydra has one copy of a Fat-like gene (HyFat) and one Dachsous (HyDs), similar to Drosophila; both genes are ubiquitously expressed in epithelial cells. Using an antibody raised against HyFat, she then demonstrated that HyFat protein is distributed in a polarized fashion on the apical side of body column epithelium, suggesting a conserved role for HyFat in PCP establishment. In support of this, she demonstrated that knocking down HyFat resulted in severely deformed head structures. Tapan Goel  shared his fascinating story of Hydra mouth opening dynamics. Hydra tear their hypostome tissue every time they open their mouth. It is a process driven by cell shape changes with little or no cell rearrangement [Hydra mouth opening process. He then discussed a series of perturbations he is performing, including using nerve-less Hydra and laser ablation of actin myonemes, with the goal of understanding the biology underlying his observations. In the final talk of the Cell Biology session, Taylor Skokan  showed stunning live imaging of transgenic Hydra expressing Lifeact-GFP in ectodermal epithelial cells which revealed surprising actin dynamics. He observed large ring-shaped protrusions being formed on the apical surface of epithelial cells with extraordinary dynamics, reminiscent of macropinosomes found in mammalian macrophages. The formation of these structures can be robustly induced by the addition of Gd3+ ions and can be inhibited by applying pressure from the coverslip. The physiological purpose of these structures remains unknown, but this study nicely showcased the potential for application of advanced microscopy in cnidarian research.The relative simplicity of the cnidarian tissue architecture permits interrogation of cell biological processes that are fundamental to development and regeneration. In recent years, comparative analysis using cnidarian systems has provided new, and often surprising, insights into the diverse cellular programs employed during morphogenesis as well as tissue homeostasis –46. Hele systems –49. McNeangement . Goel noHydractinia allorecognition, the process by which colonies decide whether to fuse or fight when they come into contact [Alr1 and Alr2, were previously identified as key players. Through careful observation, Huene realized that Alr1 and Alr2 alone are insufficient to explain all outcomes when two Hydractinia colonies interact. Colonies sharing identical Alr1/Alr2 loci sometimes exhibit partial fusion, suggesting the presence of additional determinants. In collaboration with the Schnitzler and Baxevanis groups, Huene identified over 60 Alr-like genes within the same genetic complex near Alr1 and Alr2. Through a series of genetic recombination experiments using outbred colonies, she mapped a third allodeterminant onto a 0.4-Mb genomic region. These results demonstrate that Hydratinia allorecognition relies on a more sophisticated molecular program than previously thought. Nikki Traylor-Knowles (University of Miami) presented progress in understanding the immune response of the coral Pocillopora damicornis. With collaborators, she sequenced and analyzed the genome and found that immune-related genes, such as components of the NF-κB pathway, were expanded in Pocillopora. She found that some immune-related genes are expressed in nematocytes, echoing recent findings in Nematostella [Nematostella. TLRs are a family of transmembrane receptor proteins that are capable of pathogen recognition in bilaterian animals. Nematostella possesses a single TLR gene that is structurally similar to the orthologous vertebrate gene. Using tissue culture systems, Williams first showed that NvTlr is able to activate the NF-κB pathway in human cell lines by recognizing heat-inactivated coral pathogen Vibrio coralliilyticus and bacterial flagellin protein. Next, using in situ hybridization and immunofluorescence, she found that nematosomes (motile bodies specific to Nematostella) are highly enriched for NvTlr and NF-κB components. Moreover, she demonstrated that nematosomes are capable of engulfing fluorescently labeled V. coralliilyticus. Genetic perturbations of NvTlr and NvNF-κB by morpholinos elicited developmental defects as well as failure of nematocyte specification. Taken together, these results suggest that innate immune signaling is functionally conserved in Cnidaria.Cnidarians have mechanisms to defend against microbial pathogens and distinguish between self and non-self, while often maintaining a beneficial symbiotic relationship with dinoflagellate symbionts , 52. A d contact , 23, 53.tostella . FinallySymbiodiniaceae. Reef-building corals, for instance, cannot thrive in nutrient-poor tropical waters without nutritional assistance from their phototrophic symbionts. As emphasized by Weis in the keynote address, understanding the cnidarian–dinoflagellate symbiotic relationship is an urgent task, as global warming and seawater acidification are threatening the existence of coral species all over the world. The small sea anemone Aiptasia is widely used in laboratories as a model system to study cnidarian–symbiont interactions because unlike corals, Aiptasia can be easily cultured and manipulated in a laboratory setting. Lorraine Ling  investigated the role of C-type lectins in host–symbiont recognition using Aiptasia. C-type lectins are a group of carbohydrate-binding proteins that facilitate cell–cell adhesion, immune response, and pathogen detection. Ling found that two C-type lectins, Ctl1 and Ctl6, are downregulated during symbiont establishment. Next, using in vitro binding assays, she found that Ctl1 recombinant protein binds strongly to incompatible strains, indicating that C-type lectins might contribute to the specificity of host–symbiont recognition. Thomas Gilmore (Boston University) focused on the dynamics of the NF-κB pathway during symbiont establishment in Aiptasia. Gilmore found that NF-κB signaling activity increases upon depletion of symbionts and decreases upon introduction of compatible symbionts to naïve Aiptasia. Symbiotic Aiptasia were more susceptible to pathogenic bacterium, which may explain why lower levels of bacterial infection were found in recently bleached corals. Taken together, these data support the hypothesis that symbionts partially suppress the host immune system and may compromise the ability of the host to respond to foreign pathogens or environmental stress. Casandra Newkirk  investigated the potential connection between symbiont establishment and life stage transition in the upside-down jellyfish, Cassiopea xamachana. The acquisition of algal symbionts at the polyp stage is a prerequisite for the metamorphosis and subsequent strobilation. Newkirk generated asymbiotic polyps and tested the ability of different strains of symbionts to grow in the polyp and trigger strobilation. Among the three strains tested, strain A194 exhibited the slowest growth and slowest trigger for strobilation. However, the timing of strobilation did not correspond to the absolute number of symbionts within a host polyp, indicating the presence of additional signals. The final speaker of the session, Juris Grasis , argued that viruses, just like bacteria and eukaryotic symbionts, are an inseparable component of the animal holobiont. Using Hydra as a model system, Grasis surveyed the Hydra virome with sequencing methods [Hydra-associated viruses and their function during host immune response. Interestingly, Grasis observed a strong immune response when introducing foreign viruses into Hydra, even under germ-free conditions, suggesting that Hydra possess an underappreciated viral discrimination mechanism.Many cnidarian species, especially corals and sea anemones, host intracellular symbionts: dinoflagellates of the family  methods . Taking Nematostella. Previous studies have demonstrated the existence of behavioral, transcriptional, and metabolic rhythmicity in Nematostella polyps subjected to diel light cycle [NvPar-bZIPC, stopped cycling in response to light removal while others, such as NvCry1a, remain oscillating. Interestingly, even at 48 h post-light removal, oscillation patterns were observed in approximately 180 different genes, suggesting the presence of a persistent circadian rhythm. Hanny Rivera  used Nematostella to better understand the genetic inheritance of thermal tolerance. She found that Nematostella populations in the wild are subjected to a large range of daily temperature fluctuations. She therefore tested in the laboratory the thermal tolerance of Nematostella planula larvae produced from parents reared in two different temperature conditions: (1) constant temperature and (2) intermittent heat shock. She observed a possible inheritance of thermal tolerance that may be transmitted maternally. Finally, Rivera proposed to test Nematostella strains collected from different geological locations to better understand the inheritance and adaptation of thermal tolerance in wild populations. Sérgio Stampar (São Paulo State University) discussed the advantages of using the traditionally understudied tube anemone, Ceriantharia, as a model system to study the influence of the environment on animal life stages. By refining the life stage descriptions of Ceriantharia, Stampar and his collaborators revealed remarkable diversity and plasticity in the development of these animals, such as the presence or absence of a planula larvae stage. Depending on nutritional condition and additional environmental cues, a single Ceriantharia species can remain as a planktonic larva for a variable amount of time, ranging from a week to 3 months. This extreme flexibility during development, in addition to other unique biological traits, such as a linearized mitochondrial genome and abnormal toxin assemblage, makes Ceriantharia an attractive model system for future cnidarian research [Pocillopora corals. She performed a screen to identify biomarkers that could be used to isolate coral cells by FACS. She found that coral cell types had differential binding affinities to certain dyes and that autofluorescence from dinoflagellate symbionts could be used to isolate gastrodermal cells.Cnidarians, such as corals and sea anemones, form the foundation for some of the most diverse and complex ecosystems in the world. Exploring the interactions between cnidarians and their living habitat provides crucial information to help ensure their survival in the everchanging environment. Whitney Leach  explored the molecular nature of the circadian clock in ht cycle , 56–58. research . FinallyCordylophora caspia moved from a strictly estuarine environment to freshwater. Multiple subspecies tolerate different salinity ranges, presenting a perfect opportunity to investigate the marine to freshwater transition. Using RAD-seq methods, Chang built a phylogeny of Cordylophora collected from different geological locations with different salinities. By mapping the native salinity on to the phylogenetic tree, she demonstrated that lineages are separated by salinity. Ancestral character state reconstruction revealed that the Cordylophora ancestor may already have possessed the ability to tolerate freshwater. Adolfo Lara  presented his work on the phylogenetic distribution of gap junction proteins in Cnidaria. By surveying multiple cnidarian species, including understudied branches such as Staurozoa, Myxozoa, and Cubozoa, Lara found that pannexins are enriched in anthozoan species such as Nematostella, while innexins are exclusively found in medusozoans. How this impacts the function of gap junctions in these clades requires further investigation. Meg Daly (Ohio State University) presented her work exploring the evolutionary relationships within the anthozoan lineage. She first determined the relationships between major anthozoan lineages using highly conserved DNA sequences, and subsequently relied on variable genes to determine relationships within each lineage. To add additional species in the absence of genomic data, Daly used morphological analysis to manually determine position on the tree. Her phylogeny suggests that the presence of symbionts is a rare trait in anthozoans that independently evolved multiple times. Last, Lauren Vandepas  analyzed the evolution and function of chitin synthase across Cnidaria. Taking a genomics approach, Vandepas identified chitin synthase genes in several cnidarian genomes and using an affinity tag for chitin she found that chitin is indeed present in the tissues of scyphozoan, hydrozoan, and anthozoan animals. In Nematostella, for instance, chitin is found in spirocytes (a type of stinging cell) and in the mesoglea. Chitin is a common biomolecule that provides structural support for animals such as arthropods and crustaceans, but a function in cnidarians is unexplored. Vandepas proposed that cnidarians might use chitin to provide additional structural support.As a sister group to bilaterians, cnidarians are keys to understanding evolutionary transitions, such as segregation of the mesoderm, bilateral symmetry, and nervous system centralization. Cnidarians share many conserved genes and signaling pathways with bilaterians, so these transitions must largely result from differential use of the same genetic toolkit. Natasha Picciani  presented her recent findings regarding the evolution of eyes in cnidarians . Many cnNematostella vectensis; Paige Zhang  probed the DNA methylation pattern of the moon jelly, Aurelia sp.; Hiroshi Shimizu (King Abdullah University of Science and Technology) presented a potential gravity sensing mechanism in Hydra; Bryan Teefy  discussed the function of the PIWI-piRNA pathway in Hydra to repress transposons in somatic stem cells.A total of 43 posters were displayed in two poster sessions during the meeting. Before each poster session, all poster presenters gave a 2-min lightning talk to advertise their research to all meeting attendees. According to post-meeting survey results, the lightning talks were effective and well received. The posters covered diverse topics of cnidarian biology, including genomics and transcriptomics, regeneration and stem cell biology, behavioral biology, neuroscience, ecology, and evolution. Due to limitations of space, we will not be able to discuss all of the exciting work presented during the poster sessions. A few highlights include: Bob Zimmermann  presented the community effort to generate a chromosome-level genome assembly of the starlet sea anemone Among the many outstanding presentations and posters, Ahmet Karabulut  and Taylor Skoken  won the best oral presentation awards; Jennifer Spillane  and Shelcie Menard  won the best lighting talk awards; and Abby Primack  and Rui Wang  won the best poster awards.As emphasized by Virginia Weis in her keynote address, it takes a village. It takes community effort, not only to advance coral symbiosis studies, but also to push forward the entire field of cnidarian research. At this meeting, the emergence of a bright new generation of cnidarian biologists was on display and is evidence that our community is growing. The development of new technologies that can be readily applied to cnidarians is flooding our field with new and exciting ideas. This US-based meeting will be held on a biennial basis, alternating between east and west coasts to balance travel burdens. The next meeting will be held at the University of California, Davis on September 9–13th, 2020.


Telomere crisis is linked with many of the genomic alterations found in cancer genomes. A new understanding of how these alterations arise points towards an active role for innate immune sensors during crisis and to new opportunities for the treatment and diagnosis of cancer. Telomeres protect genomic integrity by masking natural chromosome ends from the DNA damage response and repair pathways. The erosion of telomeric sequence due to incomplete replication compromises this protection with broad consequences for aging. In the context of cancer, telomere shortening can exert a tumor-suppressive effect by enforcing a proliferation arrest. On the other hand, telomere deprotection can also enable cancer growth through telomere crisis, a state of extensive genomic instability and cell death. Telomere crisis causes numerous, cancer-relevant genome alterations, including translocations, amplifications, and deletions, and has now been linked with the genesis of the mutational phenomena chromothripsis and kataegis . ChromotDuring telomere crisis, the aberrant activation of DNA repair pathways at natural chromosome ends results in telomere–telomere fusion and the creation of dicentric chromosomes Fig. a. AlthouNuclear envelope rupturing may be an important driver of genomic alteration during telomere crisis. Outside of telomere crisis, nuclear envelope rupturing occurs in micronuclei, aberrant nuclear structures formed after errors in cell division, where it has been linked with DNA damage and chromothripsis , 5. PrimThe induction of APOBEC3 mutagenesis is another threat to genomic integrity during telomere crisis. The APOBEC3s are a family of cytosine deaminases that specifically target single-stranded DNA as part of their normal function as anti-viral restriction factors. An APOBEC3-linked mutational signature is found in at least 22 cancer types, in which it can occur at high frequency. APOBEC3 mutations often cluster at rearrangement breakpoints, where they are termed kataegis clusters, or they can be dispersed throughout the genome. Observations to date suggest that APOBEC3 mutagenesis during telomere crisis is limited to kataegis clusters. The cause of APOBEC3 dysfunction during crisis is not known but may be related to the observed accumulation of single-stranded DNA at DNA bridges . LikewisTrex1 gene cause Aicardi-Goutières syndrome, which is characterized by elevated type I interferon levels and severe encephalitis. These symptoms have been linked with activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) cytosolic DNA-sensing pathway, which detects cytosolic DNA and triggers a wide-ranging, anti-viral response that includes the induction of type I interferons and other pro-inflammatory genes. The cGAS-STING pathway senses and responds to cytosolic DNA species that accumulate as a result of genomic instability by activating an IRF3- and NFκB-dependent pro-inflammatory, transcriptional response. This inflammatory response can induce senescence and even apoptosis and thus have pronounced effects on cancer cells. The full impact of this inflammatory response in a clinical setting is not known, but there is evidence that it can influence the efficacy of radio- and immunotherapies. We speculate that the cGAS-STING pathway promotes replicative senescence and limits escape from telomere crisis. In support of this view, cGAS, the DNA-sensing component of this pathway, has been previously observed at DNA bridges [It is likely that TREX1 is not the only cytosolic factor to engage genomic DNA during telomere crisis. Loss of function mutations in the  bridges .Genomic rearrangements that are induced during telomere crisis may contribute to carcinogenesis by driving genetic change, but these alterations cannot accumulate indefinitely because they would eventually inhibit cancer growth. Escape from telomere crisis requires re-activation of the telomerase reverse transcriptase (TERT), which is normally silenced during development. Telomerase can enable escape from telomere crisis by synthesizing telomeric repeats de novo at chromosome ends, thus healing shortened telomeres and restoring the capacity for DNA proliferation.The identification of activating mutations in the TERT promoter point towards one major mechanism of telomerase reactivation. MSK-IMPACT, a large-scale clinical sequencing initiative, has identified these mutations as the most frequent non-coding mutations in cancer . DespiteA recent analysis of clear cell renal carcinomas has identified three additional frequently appearing mutations, which are independent of the highly recurrent TERT promoter mutations, in the TERT 5′ untranslated region  Fig. 1bb1b. The Telomere crisis is associated with a nearly comprehensive list of genomic alterations. Progress in the field will require mechanistic work to determine the sources of DNA damage during crisis and how these sources contribute to distinct genomic consequences. The data point towards nuclear envelope rupturing as a significant mediator of instability during crisis. In the future, it will be interesting to determine whether the DNA damage that results from nuclear envelope rupturing at DNA bridges, micronuclei, and the nucleus occurs through distinct or similar mechanisms in each of these locations.In addition to this hypothesized role in genomic rearrangement, nuclear envelope failure at DNA bridges may prove to be significant during telomere crisis as the result of its engagement of other components of the cGAS-STING signaling pathway in addition to TREX1. cGAS and STING have pivotal roles in cancer immunity and in the anti-tumor effects of immune checkpoint blockade. With this in mind, cGAS-STING activation by DNA bridges may promote cancer immunogenicity. We speculate that this pathway could boost the anti-tumor benefits of therapies targeting telomere maintenance. Future work will determine the extent of cGAS-STING activation during telomere crisis and how this activation impacts cell viability, escape from crisis, immune engagement, and potential therapies.More studies will be required to identify additional mechanisms driving telomerase reactivation in cancer. Telomerase promoter mutations are not sufficient on their own to indefinitely delay replicative senescence and are proposed to work as part of an uncharacterized two-step mechanism . LikewisCollectively, these recent advances have set the stage for deep insights into how telomere crisis shapes the cancer genome and engages with cytosolic DNA-sensing pathways to alter the course of disease.


Sclerostin is a well-known inhibitor of bone formation that acts on Wnt/β-catenin signaling. This manuscript considers the possible role of sclerostin in vascular calcification, a process that shares many similarities with physiological bone formation. Rats were exposed to a warfarin-containing diet to induce vascular calcification. Vascular smooth muscle cell transdifferentiation, vascular calcification grade, and bone histomorphometry were examined. The presence and/or production of sclerostin was investigated in serum, aorta, and bone. Calcified human aortas were investigated to substantiate clinical relevance. Warfarin-exposed rats developed vascular calcifications in a time-dependent manner which went along with a progressive increase in serum sclerostin levels. Both osteogenic and adipogenic pathways were upregulated in calcifying vascular smooth muscle cells, as well as sclerostin mRNA and protein levels. Evidence for the local vascular action of sclerostin was found both in human and rat calcified aortas. Warfarin exposure led to a mildly decreased bone and mineralized areas. Osseous sclerostin production and bone turnover did not change significantly. This study showed local production of sclerostin in calcified vessels, which may indicate a negative feedback mechanism to prevent further calcification. Furthermore, increased levels of serum sclerostin, probably originating from excessive local production in calcified vessels, may contribute to the linkage between vascular pathology and impaired bone mineralization. Cardiovascular disease is responsible for a substantial part of mortality among the elderly and patients with diabetes, chronic kidney disease (CKD), hypertension, and osteoporosis. Vascular media calcification  is a prominent aspect of cardiovascular disease in these patient populations. Vascular calcification is an active cell-regulated process in which the vascular smooth muscle cells (VSMCs) play a prominent role . One of the key events during the vascular calcification process is the transdifferentiation of VSMCs, a process that now potentially affords the capacity for being exploited as an important therapeutic target . Since tIn general, the precise role of sclerostin in VSMC transdifferentiation and the development of vascular calcifications, as well as its potential role in the bone–vascular axis, is poorly understood. This issue should be of particular clinical interest given the recent therapeutic introduction of an antisclerostin antibody treatment to stimulate bone formation . Since cTo investigate our theory that sclerostin, produced by VSMCs in calcified vessels, may spill over to the serum and thereby, next to impacting the bone, prevent further progression of (vascular) calcifications, a rat warfarin model was employed. In contrast to other models of vascular calcification , this model induces vascular calcifications in the absence of underlying bone pathologies. Hence, the disturbed mineral metabolism in CKD, diabetes, and osteoporosis patients  seriously complicates the mechanistic understanding of bone–vascular interactions. In our experimental subjects, warfarin exposure prevented the production of biologically active matrix γ-carboxyglutamic acid protein (MGP) ,20. MGP Induction of vascular calcification in rats by dietary exposure to warfarin resulted in low overall mortality of 6.7%. Two warfarin-exposed rats died, out of which one animal died 40 days and the other animal 2 days before the planned sacrifice at week 10 . Body weight; food intake; and serum calcium, phosphorous, and creatinine were compared between control and warfarin-exposed rats at week 10 and did not differ between control and warfarin-exposed rats . Serum cp < 0.05) differential regulation of 275 proteins  in the aorta. Using latent semantic analysis (LSA)-based interrogation of this list of 275 proteins, employing either Wnt/Catenin or PPAR as the investigating concept, we identified a strong representation in this dataset of proteins linked to both osteogenic (Wnt/Catenin) and adipogenic (PPAR) transdifferentiation. To study VSMC transdifferentiation, relative quantitative proteomic analysis of aortas extracted from control rats and rats exposed to warfarin for 10 weeks was performed. Using stringent and significant cut-off criteria for protein identification (99th percentile peptide identification confidence), we found that warfarin exposure caused significant  compared with control animals.Sclerostin levels gradually increased in a time-dependent manner in the serum of rats exposed to warfarin for 4, 6, 8, and 10 weeks . At weekSost) mRNA levels were significantly upregulated  in warfarin-exposed rats compared with control rats at week 10 (Aortic sclerostin ( week 10 . p = 0.0095) in rats exposed to warfarin compared with control rats. Measures of osteoblast activity (bone formation), including osteoblast perimeter and osteoid area, were lower (in a nonsignificant manner) in warfarin-exposed rats compared with controls. Bone resorption (osteoclast perimeter) and bone mineralization  did not differ between control and warfarin-exposed rats.The analysis of static and dynamic bone parameters in control and warfarin-exposed (10 weeks) rats is presented in To investigate osteocytic sclerostin production, immunostaining for sclerostin was performed in the bone. Microscopic analysis of the staining allowed the clear distinction of sclerostin-positive from -negative osteocytes in the bone. The frequency of sclerostin-positive versus -negative osteocytes was recorded by manually counting in a label-blinded manner the osteocytes in the tibia sections. No differences in the percentage of sclerostin-positive osteocytes were observed between control and warfarin-exposed rats .Many aspects of the vascular media calcification process remain cryptic. Although being generally accepted as the key event during the development of vascular calcification, knowledge of VSMC transdifferentiation is rather fragmentary. Moreover, at this stage, it is not clear which molecules exactly are key in the linkage between bone and vascular pathology (bone–vascular axis). Since the disturbed mineral and bone metabolism in CKD patients further complicates the understanding of the mechanisms underlying the bone–vascular coupling, an experimental animal model with warfarin-induced vascular calcification was applied in this study. This model gave us the opportunity to investigate vascular calcification and the bone–vascular axis, regardless of CKD-induced disturbed mineral balance and the resulting bone pathology. Indeed, serum calcium and phosphorous levels were similar between control and warfarin-exposed rats. During the vascular calcification process, one of the key events is the transdifferentiation of VSMCs . Given tAn inverse relationship exists in the adipogenic and osteogenic lineage commitment, which may indicate that promoting the adipocyte phenotype may exert antagonistic effects on calcification, which is in line with the fact that PPARγ signaling counteracts vascular calcifications ,4,23 andIn our current study, we found that sclerostin was expressed at both the mRNA and protein level in calcified vessels, which is in line with earlier studies ,11,24. FOsteocytic sclerostin production was investigated to ensure that the elevated serum sclerostin levels were due to sclerostin production by calcified vessels and did not originate from the osteocytes, which are the main source of sclerostin. Our finding that no differences were observed in the number of sclerostin-expressing osteocytes between control and warfarin-exposed rats strongly indicates that the bone is not likely to be responsible for the observed increased serum levels of sclerostin. Furthermore, we recently found sclerostin protein in the cell culture medium of in vitro calcifying VSMCs, proving that VSMCs should be able to secrete sclerostin towards the circulation (unpublished results).Bone histomorphometric analysis of warfarin-exposed animals revealed a mildly, however significantly decreased  bone area after 10 weeks of warfarin exposure compared with control rats. This finding is in line with the decreased  number of osteoblasts (osteoblast perimeter) and reduced (not significant) amount of associated osteoid material found. Mineralization of the deposited osteoid, however, remained intact, as reflected by the absence of any difference in osteoid width and mineral apposition rate. As indicated by the osteoclast perimeter, bone resorption also did not differ between groups. Given the particular role that has been ascribed to sclerostin at the level of the bone , the ratThis study has a few limitations. Since this study was originally designed to only assess the degree of vascular calcification due to warfarin treatment, no urine was collected. Being able to measure the creatinine content in the urine would have made it possible to calculate the glomerular filtration rate (GFR), which is based on the measurement of urine and serum creatinine concentrations and the urinary volume, this is a more accurate measure of kidney function compared with serum creatinine concentration. Future experiments are needed to investigate to what extent increased renal production of sclerostin, in addition to calcified VSMC, contributes to increased serum levels of this protein. Overall, this study demonstrated that the sclerostin biology in an animal model of vascular calcification is in agreement with the role of this protein in the vascular calcification process and the pathologically disturbed bone–vascular axis in patients suffering from vascular calcifications. Similar to its function in bone, it could be assumed that sclerostin is produced as a negative feedback mechanism to prevent excessive calcification. These results urge the investigation of the vascular safety of antisclerostin antibody treatment, which currently is ongoing in our laboratory. Hence, antisclerostin treatment is being developed as a bone anabolic treatment for patients with osteoporosis, who are at the same time at risk for vascular calcifications. All experimental procedures were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals 85-23 (1996) and approved by the University of Antwerp Ethical Committee for Animal Experiments. The experimental procedure involving human arterial sections was approved by the ethics committee of the University of Antwerp (A03 043) on 18 December 2014.Thirty 8-week-old male Wistar rats  were housed in standard cages (two per cage) at constant temperature and humidity and exposed to a 12 h light/dark cycle. All rats had free access to tap water and their assigned diet. The animals were randomly assigned to the following experimental groups: control group (10 weeks (wk), n = 4) and warfarin-exposed groups . Control rats were fed standard pellet chow for 10 weeks. Rats receiving the warfarin-containing diet 3 to prevent chemical interference. Serum phosphorus levels were measured using the Ecoline®S Phosphate kit . At sacrifice, rats were exsanguinated via the retro-orbital plexus after anesthesia with sodium pentobarbital  via intraperitoneal injection. Serum creatinine was measured according to the Jaffé method. Calcium levels were determined using flame atomic absorption spectrometry  after diluting the sample in 0.1% La and MS2 level . The raw data were analyzed by Proteome Discoverer 2.0 software (ThermoScientific) using Sequest HT as search engine against the rattus norvegicus UniProt/SwissProt database with a threshold of confidence above 99% . The list of identified proteins containing iTRAQ ratios of expression levels over control samples was then generated.Mass spectrometry and quantitative proteomics (using iTRAQ labeling) were performed on the aortic samples of control rats and rats exposed to warfarin for 10 weeks. The aorta samples were ground completely in a protein extraction buffer . The concentrations of the proteins extracted from the aorta were quantified using the reducing agent and detergent compatible (RCDC) protein assay kit . Equal amounts of proteins from each sample were reduced and alkylated by tris-2-carboxyethyl phosphine and 5-methyl-methanoethiosulphate, respectively, before trypsin digestion. The resulting peptides from each sample were labeled using iTRAQ reagents  following the manufacturer’s instructions. To improve LC-MS/MS proteome coverage, samples were subjected to a 2D-LC fractionation system . The mixed peptides were first fractionated on a strong cationic exchange chromatography polysulfoethyl aspartamide column ) and secondly separated on a nano-LC C18 column ). The nano-LC was coupled online to a QExactive™-Plus Orbitrap (ThermoScientific) mass spectrometer. The nano-LC eluents were infused to the Orbitrap mass spectrometer with a capillary at 1.7 KV on a nanoelectrospray ionization (nano-ESI) source at a flow rate of 300 nL/min. Data-dependent acquisition in positive ion mode was performed for a selected mass range of 350–1800 2 transformed. Following log2 ratio transformation, differentially expressed protein (DEP) lists were created by identifying only proteins that possessed log2-transformed iTRAQ ratios two standard deviations (p < 0.05) from the calculated mean background expression variation level. Significant DEP lists (comprising proteins elevated or reduced in their expression in response to warfarin) were then employed for further bioinformatics deconvolution using diverse informatics platforms including Ingenuity Pathway. LSA of the warfarin DEP lists was performed as previously described using GeneIndexer [Proteins identified according to the statistical MS cut-offs described previously were then subsequently used for bioinformatic analyses. To identify the significantly altered proteins , raw iTRAQ ratios (control:warfarin) were first logeIndexer . LSA is At sacrifice, aorta, left femoral, and left carotid arteries were isolated. For histological analyses, thoracic aortae were fixed in neutral buffered formalin for 90 min and cut into 2–3 mm thick rings. These rings were then embedded upright in paraffin, of which 4 μm sections were cut and stained for calcification with von Kossa and counterstained with hematoxylin and eosin (H&E). Calcification was evaluated using Axiovision image analysis software . Two color thresholds allowed separation of the calcified area  and the remaining tissue area. The percentage calcified area is defined as the von Kossa positive area versus the total tissue area (von Kossa positive area plus the remaining tissue area).3 at 60 °C, followed by dilution in 0.1% La(NO3)3 to eliminate chemical interference during flame atomic absorption spectrometry. Results were expressed as mg calcium/g wet tissue.To determine the calcium content, the proximal part of the abdominal aorta, the left carotid, and femoral arteries were weighed on a precision balance. The tissue samples were then digested overnight in 65% HNOTo allow histomorphometric analysis of dynamic bone parameters, all animals received an intraperitoneal injection of 30 mg/kg tetracycline and 25 mg/kg demeclocycline 7 and 3 days before sacrifice, respectively. At sacrifice, the left tibia was isolated and fixed overnight in 70% ethanol. After dehydration and embedding in 100% methyl methacrylate , 5 µm thick sections were stained by the method of Goldner for quantitative histology to determine static bone parameters using Axiovision image analysis software . Total bone area, mineralized bone area, osteoid width and area, osteoblast perimeter, eroded perimeter, osteoclast perimeter, and trabecular number were measured using this software platform. Unstained sections of the tibia (10 µm thick) were mounted in 100% glycerol for fluorescence microscopy and visualization of tetracycline and demeclocycline labels. Based on the length and distance between double tetracycline/demeclocycline labels, dynamic bone parameters, including bone formation rate and mineral apposition rate, were measured. Total mRNA was extracted from the distal part of the abdominal aorta using the RNeasy Fibrous Tissue Mini Kit  and complementary DNA was generated with the High-Capacity cDNA Archive Kit . Real-time PCR amplification was performed based on the TaqMan fluorescence methodology . TaqMan probes and primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Rn99999916_s1) and sclerostin (SOST) (Rn00577971_m1) were purchased from Applied Biosystems as a Taqman® gene expression assay on demand. For each sample, the expression of the tested transcripts was analyzed in triplicate and normalized to the expression of GAPDH, the housekeeping gene. The comparative CT method, using aortas of control rats as calibrator samples, was used to calculate the gene expression levels.Immunohistochemical staining was performed on neutral buffered formalin-fixed, deparaffinized arterial sections of warfarin-exposed and control rats sacrificed at week 10, as well as on calcified human arterial sections. The excised tissue sections were blocked with normal goat serum (20% in PBS) for 20 min and incubated overnight with polyclonal rabbit antisclerostin  or LRP4 . Biotinylated goat anti-rabbit  was used as a secondary antibody. Avidin/biotinylated peroxidase complex  was added as a signal amplifier and 3-amino-9-ethylcarbazole  was used as a substrate. The sections were counterstained with hematoxylin. Sections in which the primary antibody was omitted were used as negative controls. Sclerostin expression was also investigated on tibia sections in order to compare the absolute sclerostin expression per bone area as the percentage of sclerostin-positive osteocytes (lacunae) between control and warfarin-exposed rats. After deacrylation and decalcification of the tibia sections, the same protocol for immunohistochemistry on the arterial sections was used as described above. The antisclerostin primary antibody  was used in a 1:750 concentration. The sclerostin concentration in the serum was determined using an antisclerostin ELISA kit , following the appropriate procedures.U test. Bonferroni correction was applied when appropriate. p-Values < 0.05 were considered statistically significant. All statistical analyses were performed using PRISM software . Data are presented as the mean ± standard error of mean (SEM). Statistical differences between groups were investigated by a two-tailed Mann–Whitney


COI barcoding. The aim of this longitudinal study was to evaluate AIV dynamics in a natural wetland ecosystem, taking into account both virological aspects and ecological traits of hosts. Global AIV prevalence decreased significantly during the second sampling period (0.3%) compared to the first (6.6%). Circulating subtype distributions were also different between periods, with a noteworthy H5 and H7 subtype richness during the first sampling period. Mallard Anas platyrhynchos was identified as the main AIV host, although not all positive samples could be ascribed to the host. We modelled AIV prevalence with regard to the avian host community composition and meteorological data from the wetland. Statistical analysis revealed seasonal differences in AIV detection, with higher prevalence during the breeding season compared to other phenological events. The model also shows that the lower AIV prevalence during the second study period was associated with a significant reduction of breeding Anseriformes in the wetland, revealing a long-term fluctuation of AIV prevalence driven by the breeding Anseriformes community. This longitudinal study on AIV epidemiology in a natural ecosystem reveals that although prevalence follows seasonal and annual patterns, long-term prevalence fluctuation is linked to the breeding community composition and size. These results are relevant to understanding the influence of host ecology on pathogen transmission for preventing and managing influenza emergence.During 2007–2009 and 2012–2014, avian influenza virus (AIV) was studied in a wild avian community of a northern Spanish wetland using non-invasive sampling methods and host identification by The online version of this article (10.1186/s13567-019-0623-5) contains supplementary material, which is available to authorized users. Orthomyxoviridae, genus Influenzavirus A) owe their diversity to the antigenic properties of two surface glycoproteins [16 known haemagglutinin variants (HA) and 9 neuraminidases (NA)], that combined lead to 144 potential HA/NA subtypes . Hence, all roosting waterbird species were regarded as potential hosts and interspecies natural mixing was considered. Special attention was paid to circulating viral strains, to host species harbouring AIV and to ecological factors with a potential effect on viral detection.The aim of this longitudinal study was to evaluate AIV dynamics in a natural wetland ecosystem, by taking into account virological aspects and ecological traits of hosts during two different sampling periods. Our sampling strategy was based on non-invasive sampling methods, which consisted of environmental fresh faecal sample collection after Salburua wetland  is a 217.46 ha wetland area located in Basque Country that pertains to the “Northern-Plateau” bioregion of Spain . Under tWetland of International Importance Ramsar designation in 2002 and Site of Community Importance in 2004 within the European Natura 2000 Network. From an ornithological perspective, the wetland is strategically situated, as many bird species use these lagoons for wintering, breeding or for stopover along the East Atlantic flyway while migrating. The bird community on Salburua wetland includes mallards, coots (Fulica atra), Northern shovelers (Anas clypeata), white storks (Ciconia ciconia), gadwalls (Mareca strepera), common teals (Anas crecca), common pochards (Aythya ferina), common moorhens  crested grebes (Podiceps cristatus) and little grebes (Tachybaptus ruficollis), with varying numbers depending on the season, especially for migratory species. Since 2005, when monitoring plans for AIV began in the Basque Country, this wetland has had a frequent LPAIV record and the only HPAIV H5N1 wild bird case in the Iberian Peninsula so far .Host identification was successful in 48% (25/52) of the AIV-positive samples , breeding grebe couples (Podiceps cristatus), breeding little grebe couples (Tachybaptus ruficollis), breeding Anseriformes, and breeding non-Anseriformes/non-Charadriiformes  in a small wetland in northern Spain and adds to the still scarce information on long-term AIV dynamics. The present work is a continuation to a short-term cross-sectional study that characterised several different wetlands in parallel. That study, compared factors that modulated LPAIV prevalence in a particular wetland and showed that location and climate are more important than  the size and composition of the waterfowl community . Here, wPrevious studies have emphasised the pivotal role of the Anseriformes in AIV epidemiology, especially the mallard in the Northern Hemisphere , 56. TheIn fact, significant seasonal differences in AIV prevalence were consistently found in our longitudinal study with regards to host phenology. This finding confirmed the existence of seasonal patterns in the epidemiology of these viruses in wild ecosystems as stated by other authors , 51, 56.During autumn migration, migrating birds of different-origins congregate in large numbers within the same area joining the local avian community, facilitating multiple-strain AIV mixing by host-to-host transmission (within and between species) , 62. OthThus, integrating the ecology of both host and LPAIV, a likely scenario is that significantly higher AIV detection rates found during the breeding season and autumn migration are characterised by an input of hosts that are immunologically naïve into the wetland (chicks). The number of offspring is modulated by the number of breeding couples  and hence, it will have a direct impact on AIV infections (not excluding the emergence of new strains during other periods) , 56, 66.We observed seasonal and temporal variation in AIV subtype prevalence between sampling periods except for H3N8, which was detected in both sampling periods. This could reflect the influence of heterosubtypic immunity as stated for mallards in empirical and experimental studies . H3N8 waFor AIV surveillance in wild birds, the use of non-invasive sampling techniques such as fresh faeces collection has proven to be a cost-effective tool; large sample sizes can easily be collected from the ecosystem and avian species composition is not discriminated . CaptureDuring our sampling-periods no aquatic bird mortality was related to the presence of AIV. In fact, we do not know to what degree the different viral subtypes found during our samplings affect the health status or behaviour of the infected birds. Several studies suggest that LPAIV infections are not pathogenic in their natural reservoir , 63. In Understanding the influence of host ecology on pathogen transmission is particularly relevant to prevent and manage wildlife disease emergence . From thAdditional file 1.Sampling effort (grey bars) and AIV prevalence (orange bars) at each sampling time (between brackets). The yellow shaded area indicates that during 2010–2011 no samplings were conducted.Additional file 2.Summary of the stepwise model selection procedure based on Akaike Information Criteria to compare models (AICc) used to model avian influenza virus prevalence.Additional file 3.Avian community composition recorded in Salburua wetland. Mean taxonomic order counts and AIV prevalence according to host phenology. During 2010–2011 birds were counted but no samplings for AIV detection were performed.


Epidural electrical stimulation (EES) of the spinal cord has been shown to restore function after spinal cord injury (SCI). Characterization of EES-evoked motor responses has provided a basic understanding of spinal sensorimotor network activity related to EES-enabled motor activity of the lower extremities. However, the use of EES-evoked motor responses to guide EES system implantation over the spinal cord and their relation to post-operative EES-enabled function in humans with chronic paralysis attributed to SCI has yet to be described. Herein, we describe the surgical and intraoperative electrophysiological approach used, followed by initial EES-enabled results observed in 2 human subjects with motor complete paralysis who were enrolled in a clinical trial investigating the use of EES to enable motor functions after SCI. The 16-contact electrode array was initially positioned under fluoroscopic guidance. Then, EES-evoked motor responses were recorded from select leg muscles and displayed in real time to determine electrode array proximity to spinal cord regions associated with motor activity of the lower extremities. Acceptable array positioning was determined based on achievement of selective proximal or distal leg muscle activity, as well as bilateral muscle activation. Motor response latencies were not significantly different between intraoperative recordings and post-operative recordings, indicating that array positioning remained stable. Additionally, EES enabled intentional control of step-like activity in both subjects within the first 5 days of testing. These results suggest that the use of EES-evoked motor responses may guide intraoperative positioning of epidural electrodes to target spinal cord circuitry to enable motor functions after SCI. After several months of locomotor training with EES, all 4 subjects could intentionally control leg movements while supine with EES on. Additionally, EES enabled subjects to stand for prolonged periods of time. While attempting to replicate the results reported by Harkema and colleagues,22 we reported that EES enabled intentional control of lower extremity movements and standing within the first 2 weeks of EES in a subject diagnosed with motor complete paraplegia,12 and over the course of 43 weeks of multi-modal rehabilitation, he achieved independent stepping in the presence of EES.23Initial reports using epidural electrical stimulation (EES) applied to the dorsal surface of the lumbosacral spinal cord demonstrated that EES could induce tonic and rhythmic leg muscle activity both in animal models of complete SCI22 and incomplete24 paraplegia, as well as motor complete tetraplegia.25 Despite small sample sizes, these positive outcomes, combined with investigations of EES to reporting improved cardiovascular,27 respiratory,28–31 and autonomic32 function as well as improved body composition33 after SCI, demonstrate that EES holds considerable potential as a therapeutic intervention after SCI. To date, the scientific literature has described EES-enabled functions and the stimulation patterns, electrode configurations applied, and motor training regimens used enable functions.34–36Detailed descriptions of the motor functions that were enabled by EES have been reported by studies of humans with chronic, motor complete,22 However, X-ray imaging does not provide information regarding electrode proximity to spinal cord circuitry that enables function.37 Previous reports have briefly described the use of intraoperative electrophysiological monitoring to guide electrode array implantation over the lumbosacral spinal cord.22 However, the surgical approach to position the epidural electrode array, intraoperative electrophysiological techniques used to guide electrode positioning, and electrophysiological outcomes generated have yet to be described in detail. Herein, we provide a detailed description of the surgical approach with results from intraoperative electrophysiology of EES-evoked motor responses from leg muscles indicating appropriate positioning of the EES electrode array to enable motor functions, such as step-like lower extremity movement, after recovery from EES implantation.Previous investigations using EES in humans with SCI described the use of intraoperative X-ray guidance to identify vertebral levels T11–L1 as the target location for insertion of an electrode array.This study was performed under the approval of the Mayo Clinic Institutional Review Board with a U.S. Food and Drug Administration Investigational Device Exemption (IDE G150167), and all experiments were performed in accord with the regulations and guidelines of these regulatory bodies.38 were enrolled in this clinical trial (NCT02592668), and both provided written, informed consent to experimental procedures. Subject 1 was a 26-year-old male who sustained a T6 SCI during a snowmobile accident 3 years before study enrollment. Subject 2 was a 37-year-old male who sustained a T3 SCI from a fall 6 years before study enrollment. Data from subject 1 have been previously reported12; however, all data and analysis demonstrated in this report were not previously published.Two males diagnosed with a sensorimotor complete, American Spinal Injury Association Impairment Scale Grade A (AIS-A) SCI12 Briefly, subjects underwent approximately 60 motor training sessions performed over 22 weeks for approximately three sessions per week by a team of physical therapists and kinesiologists. Motor training sessions consisted of approximately 15 min of lower extremity stretching, 45 min of locomotor training on a treadmill, and 30 min of balance and task-specific strengthening exercises. All motor training activities were performed with body weight support and trainer assistance provided as needed.In order to confirm that rehabilitation alone would not result in functional recovery, both subjects performed motor training for 6 months before implantation of the EES system. A detailed description of pre-surgical motor training for subject 1 was previously reported.After general anesthesia, subjects were positioned prone using a Jackson surgical table and Mayfield head clamp. General anesthesia was maintained by intravenous delivery of propofol and fentanyl. Inhalation agents and neuromuscular blocking agents were avoided for purposes of intraoperative electrophysiological monitoring of spinal sensorimotor activity during EES trialing.A midline incision that spanned vertebrae T11–L2 was made based on palpation of anatomical landmarks and fluoroscopy. After initial exposure, surgical dissection of back musculature continued through the lumbodorsal fascia to the level of the spinous processes in an avascular plane using electrocautery. Once the spinous processes were exposed, vertebral level was confirmed intraoperatively by inserting a radio-opaque marker in proximity to the spinous process followed by fluoroscopy. Next, subperiosteal dissection of the muscle from the spinous process down to the lamina and then laterally toward the facets was performed. Then, the T11–12 and L1–2 interspinous ligaments were severed and the spinous processes of T12 and L1 were removed with a rongeur. The facet joints of both locations were preserved to ensure spinal stability. The laminae were then thinned with a 4-mm diamond drill bit and removed by rongeurs. Finally, the ligamentum flavum was dissected free from the underlying dura.A 16-contact epidural electrode array  was passed through the T12–L1 laminectomy and directed rostrally along the dorsal midline of the dura to span the T11–L1 vertebrae . InsertiAfter induction but before surgical exposure, 13-mm subdermal electrodes were placed over selected muscles of the lower limbs in a muscle to tendon recording montage. The active electrode was placed over the midpoint of the appropriate muscle with the reference electrode over the distal tendon. A ground electrode was placed in the anterior thigh.34 Once the array was inserted, minor changes in position were guided based on leg muscle intramuscular electromyography (EMG) recordings of EES-evoked motor responses. EES-evoked motor response recordings were captured during multiple EES configurations and stimulation parameters , progressing from rostral to caudal and wide field to local. Additionally, EMG recordings were captured from the paraspinal muscles to record a stimulation artifact, which acted as the sweep trigger for real-time display of EES-evoked motor responses.Recording electrodes were placed bilaterally into the rectus femoris (RF), vastus lateralis (VL), medial hamstring (MH), tibialis anterior (TA), medial gastrocnemius (MG), and soleus (SOL). These muscles were selected to assess selectivity of EES to activate proximal or distal muscle activation as well as to assess symmetry of activation between the legs.After confirmation of electrode position by electrophysiology, the electrode array was secured to the fascia and leads were tunneled subcutaneously to the right upper abdomen where a small subcutaneous pocket was made to house the lead wires. After meticulous hemostasis and copious irrigation with a bacitracin solution, the posterior wounds were closed in anatomical layers. Subjects were then positioned on the operating table in a lateral decubitus position. After prepping and draping the side and lateral abdomen, the lead wires were externalized and a pocket was made to insert the implantable pulse generator  in a pre-determined area that would least interfere with daily activities. The neurostimulator was connected to the lead wires and inserted into the subcutaneous pocket. The anterior wound was then irrigated with bacitracin solution and closed in anatomic layers.After 3 weeks of rest, subjects returned to the laboratory to record EES-evoked motor responses from leg muscles while lying supine using EES parameters that were applied during intraoperative recordings .12During the first 5 days of testing with EES, both subjects attempted to perform motor tasks in the presence of EES. Tasks included attempts to stand, control flexion/extension lower extremity movements while supine or side-lying, and move their legs in response to an audio or visual cue. All tasks were performed with trainer assistance provided as needed. These tasks were performed with the primary goal of identifying EES configurations and stimulation parameters that enabled intentional control of lower extremity motor activity.40 A range of electrode configurations and voltage intensities were tested based on past literature in combination with intra- and post-operative interpretations of EES-evoked motor responses. EES voltage intensity was increased until volitional control over leg muscle activity and movement was observed.41 Skin-surface EMG recordings were captured over the same leg muscles as intra- and post-operative EES-evoked motor response recordings. For subject 2, goniometer recordings of knee joint changes were synchronized to EMG recordings.To determine whether EES could enable intentional control of coordinated, robust leg muscle activity, subjects were positioned side lying with the top leg suspended using non-elastic nylon fabric support slings to allow gravity-neutral movement to identify EES parameters that enabled intentional control over robust rhythmic muscle activity to generate step-like movements.t-tests to compare muscle activity. Data that were not normally distributed were analyzed using the Wilcoxon signed-rank test. To determine statistically significant differences in motor activity across muscles, a one-way analysis of variance (ANOVA) was followed by a multiple comparisons test.EMG and goniometry data were collected at a sampling rate of 4 kHz  and analyzed using custom code written in MATLAB . Notch (60 Hz) and bandpass (20–1000 Hz) filters were applied to EMG recordings to reduce environmental artifacts. EES-evoked motor response latencies were calculated as the time from stimulus pulse to the onset of the motor response of each muscle; defined as the time at which the response amplitude exceeded ±3 standard deviations (SDs) from baseline EMG values during quiescent recordings. Mean and SD values were calculated from five consecutive stimuli. Root mean square (RMS) envelopes were calculated from full-wave rectified EMG using a moving window of 200 samples and an overlap of 50 samples. Area under the curve values were calculated by taking the trapezoidal integral of the calculated RMS EMG data. The Shapiro-Wilk method was used to test for normal distributation of data. For normally distributed data, statistically significant differences between motor response amplitudes were calculated using standard parametric p = 0.0002 and p = 0.0042 for left and right leg, respectively; subject 2, p = 0.0101 and p = 0.0110 for left and right leg, respectively). Stimulation through caudal electrode configurations resulted in distal muscle EES-evoked motor response amplitudes that were significantly higher than rostral positioning of active electrodes .Initial positioning of the electrode array within the T11–L1 vertebral region was performed using anatomical landmarks and fluoroscopy . ActivatStimulating midline electrodes on the array at the rostral, middle, and caudal regions resulted in voltage-dependent selective activation of all six recorded muscles . StimulaTo determine whether the array was positioned to allow symmetrical activation of bilateral leg muscles, electrodes that were midline on the array were activated. In subject 2, initial positioning of the electrode array resulted in EES-evoked motor responses that were asymmetric in response amplitude. Asymmetry was characterized by increasing motor response amplitudes in right leg muscle recordings as EES voltages increased. However, unremarkable changes in motor response amplitudes were observed in left leg muscle recordings as EES voltages were incrementally increased . In respp = 0.62; Three weeks after EES implantation, subjects were positioned supine and instructed to relax while EES-evoked motor responses were recorded using intraoperative electrode configurations. Leg muscle activity was recorded using EES voltages that were subthreshold, threshold, and suprathreshold with respect to evoked motor responses . EES-evohttp://www.liebertpub.com) shows the range of movement and muscle activity observed during these attempts. These data and video are from the fifth and second day of EES testing for Subjects 1 and 2, respectively. Without EES, subjects were unable to intentionally generate any sustained, coordinated muscle activity in their legs.When positioned side-lying with one leg suspended in a gravity-neutral position by a sling system, both subjects demonstrated the ability to intentionally initiate, maintain, and terminate EES-enabled robust, rhythmic leg muscle activity, and step-like leg movements. First, motor threshold was detected as the voltage that resulted in EMG activity, which was achieved at 3.5 V for subject 1 and 2.5 V for subject 2. In both subjects, when voltage increased, rhythmic step-like activity was enabled. As shown in 43 These scientific observations have created a large interest within the SCI community and a sense of urgency in translating EES into an effective rehabilitation tool for individuals with SCI-related deficits. As the scientific understanding of EES-enabled function continues to improve, along with the development of next-generation neurotechnology to interface with the spinal cord, the therapeutic potential of EES continues to increase. Here, we have detailed the surgical procedures of EES implantation for use as a foundation for future clinical investigations seeking to implant EES systems after SCI. We also provided the approach of using intraoperative EES-evoked motor responses to guide electrode array positioning and compared those motor response characteristics to post-surgical EES-evoked motor responses.EES-enabled return of intentional leg movement, standing, and independent stepping has been described in individuals with SCI who have performed motor training in the presence of EES.44 Intraoperative monitoring in subjects with SCI has been primarily used as a technique to identify impending or ongoing injury and to decrease the risk of spinal cord insult during surgical procedures. A systematic review has found that the procedure is sensitive and specific for detecting intraoperative injury, and a separate Monte Carlo simulation study found a 49% reduction in risk in post-operative complications.46 Here, we demonstrated the utility of intraoperative monitoring to guide EES electrode positioning for post-surgical EES-enabled motor activity in 2 subjects undergoing EES procedures.Intraoperative electrophysiological monitoring is a valuable technique for real-time assessment of nervous system health during surgery.37 Monitoring intraoperative electrophysiological responses in real time allowed for precise placement of the epidural electrode array. By applying stimuli at multiple different regions of the spinal cord using specific electrode configurations, selective activation of spinal sensorimotor networks was achieved and interpreted from EES-evoked motor response recordings. Rostral configurations preferentially activated proximal muscles, and caudal configurations activated distal muscles in both subjects . Based on the array location, the rostral, intermediate, and caudal stimulations were likely over the L1/L2, L3/L4, and L5/S1 spinal segments, respectively. These results suggest that spatially restricted stimulation ensures that localization of active electrodes on the array can activate the most proximal or distal muscles of the leg, or excite select intermediate muscles.Animal models have shown that localization of epidural electrodes relative to the level of the dorsal roots plays a critical role in determining the intensity of stimulation required to activate select spinal circuitry, and this dependency can vary at a submillimeter resolution.ects see . Targetiects see . PreviouIn conjunction with the use of spatially restricted stimulation to target specific leg motor activity, the symmetry of the responses was also investigated. In subject 2, the electrode array was initially found to be off-set to the right as determined by a symmetric electrode configuration, which generated activity in only the right leg muscles see . This asIn addition to providing real-time monitoring of the EES-evoked motor responses during surgery, intraoperative monitoring predicts post-operative evoked motor response characteristics and establishes the groundwork for optimizing EES stimulation parameters and active electrode configurations to be used during post-surgical EES-enabled motor activities see . EES-evoThe evoked potentials observed during surgical procedures and on the first day of stimulation after surgery provide considerable clarity of the functional connectivity from the dorsum of the spinal cord to ventral motor pools and downstream neuromuscular circuitry. These electrically evoked motor responses are a critical component used to assess sensorimotor spinal network activity after SCI, and they demonstrate the potential of EES to achieve effective recruitment patterns of muscle activity. Further, we demonstrate that within 5 days of testing, it is possible to achieve coordinated and robust motor output to generate leg muscle activity that can be intentionally modulated to create step-like movements see . This deAlthough the results we presented from these 2 subjects suggest that intra-operative EES-evoked motor responses foreshadow EES-enabled motor activity performance post-surgery, they do not directly demonstrate how these single evoked potentials can serve as a biomarker of the most effective stimulation parameters to perform step-like behavior. There are multiple factors that contribute to the complexity of interactions between EES and the dynamic state of sensorimotor spinal network excitability. This complex state of excitability prohibits a direct correlation of EES-evoked motor potential characteristics to EES-enabled motor functions. For example, changing the stimulation from a single pulse to a train of pulses or adjustment of stimulus frequency cannot readily define the optimal stimulation parameters for generating a desired motor function. Another factor that makes the translation from evoked potentials to motor outcomes difficult is that the responsiveness of the spinal network intraoperatively and soon after implantation is likely to change substantially as motor function improves. One of the most obvious differences when stimulating single evoked responses versus continuous stimulation is that the spinal networks activated in these two conditions are dramatically different, quantitatively and qualitatively. For example, the behavioral responses reflect the engagement of proprioception and a return of supraspinal connectivity. With our currently limited understanding of the mechanisms underlying the functional connections that generate a motor behavior, it cannot be expected that the response of the final common pathways to each motor pool would be similar to a single evoked response compared to the ensembles of proprioceptive and supraspinal inputs projecting to spinal sensorimotor networks.EES has shown promise to improve function previously lost attributed to SCI, and this study provides further evidence of the ability of EES to enable motor function in 2 subjects with motor complete SCI. Here, we demonstrate that intraoperative EMG recordings can be used to achieve adequate array positioning by spatially restricted stimulation, that similar spinal structures are stimulated intraoperatively, and post-operatively, and that stimulation of these structures can enable coordinated motor function within the first few days of EES. The surgical procedures and electrophysiological results presented lay the groundwork for future investigations using EES in humans with SCI.