A Predictive Model of Vaccine Reactogenicity Using Data from an In Vitro Human Innate Immunity Assay System.

A primary concern in vaccine development is safety, particularly avoiding an excessive immune reaction in an otherwise healthy individual. An accurate prediction of vaccine reactogenicity using in vitro assays and computational models would facilitate screening and prioritization of novel candidates early in the vaccine development process. Using the modular in vitro immune construct model of human innate immunity, PBMCs from 40 healthy donors were treated with 10 different vaccines of varying reactogenicity profiles and then cell culture supernatants were analyzed via flow cytometry and a multichemokine/cytokine assay. Differential response profiles of innate activity and cell viability were observed in the system. In parallel, an extensive adverse event (AE) dataset for the vaccines was assembled from clinical trial data. A novel reactogenicity scoring framework accounting for the frequency and severity of local and systemic AEs was applied to the clinical data, and a machine learning approach was employed to predict the incidence of clinical AEs from the in vitro assay data. Biomarker analysis suggested that the relative levels of IL-1B, IL-6, IL-10, and CCL4 have higher predictive importance for AE risk. Predictive models were developed for local reactogenicity, systemic reactogenicity, and specific individual AEs. A forward-validation study was performed with a vaccine not used in model development, Trumenba (meningococcal group B vaccine). The clinically observed Trumenba local and systemic reactogenicity fell on the 26th and 93rd percentiles of the ranges predicted by the respective models. Models predicting specific AEs were less accurate. Our study presents a useful framework for the further development of vaccine reactogenicity predictive models.

Hawaiian Treeline Ecotones: Implications for Plant Community Conservation under Climate Change.

Species within tropical alpine treeline ecotones are predicted to be especially sensitive to climate variability because this zone represents tree species' altitudinal limits. Hawaiian volcanoes have distinct treeline ecotones driven by trade wind inversions. The local climate is changing, but little is known about how this influences treeline vegetation. To predict future impacts of climate variability on treelines, we must define the range of variation in treeline ecotone characteristics. Previous studies highlighted an abrupt transition between subalpine grasslands and wet forest on windward Haleakala, but this site does not represent the diversity of treeline ecotones among volcanoes, lava substrates, and local climatic conditions. To capture this diversity, we used data from 225 plots spanning treelines (1500-2500 m) on Haleakala and Mauna Loa to characterize ecotonal plant communities. Treeline indicator species differ by moisture and temperature, with common native species important for wet forest, subalpine woodland, and subalpine shrubland. The frequency or abundance of community indicator species may be better predictors of shifting local climates than the presence or absence of tree life forms per se. This study further supports the hypothesis that changes in available moisture, rather than temperature, will dictate the future trajectory of Hawaiian treeline ecotone communities.

Mechanisms underlying interaction frequencies and robustness in a novel seed dispersal network: lessons for restoration.

As human-caused extinctions and invasions accumulate across the planet, understanding the processes governing ecological functions mediated by species interactions, and anticipating the effect of species loss on such functions become increasingly urgent. In seed dispersal networks, the mechanisms that influence interaction frequencies may also influence the capacity of a species to switch to alternative partners (rewiring), influencing network robustness. Studying seed dispersal interactions in novel ecosystems on O'ahu island, Hawai'i, we test whether the same mechanisms defining interaction frequencies can regulate rewiring and increase network robustness to simulated species extinctions. We found that spatial and temporal overlaps were the primary mechanisms underlying interaction frequencies, and the loss of the more connected species affected networks to a greater extent. Further, rewiring increased network robustness, and morphological matching and spatial and temporal overlaps between partners were more influential on network robustness than species abundances. We argue that to achieve self-sustaining ecosystems, restoration initiatives can consider optimal morphological matching and spatial and temporal overlaps between consumers and resources to maximize chances of native plant dispersal. Specifically, restoration initiatives may benefit from replacing invasive species with native species possessing characteristics that promote frequent interactions and increase the probability of rewiring (such as long fruiting periods, small seeds and broad distributions).

Scientists' warning - The outstanding biodiversity of islands is in peril.

Despite islands contributing only 6.7% of land surface area, they harbor ~20% of the Earth's biodiversity, but unfortunately also ~50% of the threatened species and 75% of the known extinctions since the European expansion around the globe. Due to their geological and geographic history and characteristics, islands act simultaneously as cradles of evolutionary diversity and museums of formerly widespread lineages-elements that permit islands to achieve an outstanding endemicity. Nevertheless, the majority of these endemic species are inherently vulnerable due to genetic and demographic factors linked with the way islands are colonized. Here, we stress the great variation of islands in their physical geography (area, isolation, altitude, latitude) and history (age, human colonization, human density). We provide examples of some of the most species rich and iconic insular radiations. Next, we analyze the natural vulnerability of the insular biota, linked to genetic and demographic factors as a result of founder events as well as the typically small population sizes of many island species. We note that, whereas evolution toward island syndromes (including size shifts, derived insular woodiness, altered dispersal ability, loss of defense traits, reduction in clutch size) might have improved the ability of species to thrive under natural conditions on islands, it has simultaneously made island biota disproportionately vulnerable to anthropogenic pressures such as habitat loss, overexploitation, invasive species, and climate change. This has led to the documented extinction of at least 800 insular species in the past 500 years, in addition to the many that had already gone extinct following the arrival of first human colonists on islands in prehistoric times. Finally, we summarize current scientific knowledge on the ongoing biodiversity loss on islands worldwide and express our serious concern that the current trajectory will continue to decimate the unique and irreplaceable natural heritage of the world's islands. We conclude that drastic actions are urgently needed to bend the curve of the alarming rates of island biodiversity loss.

Pre-clinical development of a novel CD3-CD123 bispecific T-cell engager using cross-over dual-variable domain (CODV) format for acute myeloid leukemia (AML) treatment.

Novel therapies are needed for effective treatment of AML. In the relapsed setting, prognosis is very poor despite salvage treatment with chemotherapy. Evidence suggests that leukemic stem cells (LSCs) cause relapse. The cell surface receptor CD123 is highly expressed in blast cells and LSCs from AML patients and is a potential therapeutic target. CD123 cross-over dual-variable domain T-cell engager (CD123-CODV-TCE) is a bispecific antibody with an innovative format. One arm targets the CD3εδ subunit of T-cell co-receptors on the surface of T cells, while the other targets CD123 on malignant cells, leading to cell-specific cytotoxic activity. Here, we describe the preclinical activity of CD123-CODV-TCE. CD123-CODV-TCE effectively binds to human and cynomolgus monkey CD3 and CD123 and is a highly potent T-cell engager. It mediates T-cell activation and T-cell-directed killing of AML cells in vitro. In vivo, CD123-CODV-TCE suppresses AML tumor growth in leukemia xenograft mouse models, where it achieves an effective half-life of 3.2 days, which is a significantly longer half-life compared to other bispecific antibodies with no associated Fc fragment. The in vitro safety profile is as expected for compounds with similar modes of action. These results suggest that CD123-CODV-TCE may be a promising therapy for patients with relapsed/refractory AML.

Bridging Computational Vaccinology and Vaccine Development Through Systematic Identification, Characterization, and Downselection of Conserved and Variable Circumsporozoite Protein CD4 T Cell Epitopes From Diverse Plasmodium falciparum Strains.

An effective malaria vaccine must prevent disease in a range of populations living in regions with vastly different transmission rates and protect against genetically-diverse Plasmodium falciparum (Pf) strains. The protective efficacy afforded by the currently licensed malaria vaccine, Mosquirix™, promotes strong humoral responses to Pf circumsporozoite protein (CSP) 3D7 but protection is limited in duration and by strain variation. Helper CD4 T cells are central to development of protective immune responses, playing roles in B cell activation and maturation processes, cytokine production, and stimulation of effector T cells. Therefore, we took advantage of recent in silico modeling advances to predict and analyze human leukocyte antigen (HLA)-restricted class II epitopes from PfCSP - across the entire PfCSP 3D7 sequence as well as in 539 PfCSP sequence variants - with the goal of improving PfCSP-based malaria vaccines. Specifically, we developed a systematic workflow to identify peptide sequences capable of binding HLA-DR in a context relevant to achieving broad human population coverage utilizing cognate T cell help and with limited T regulatory cell activation triggers. Through this workflow, we identified seven predicted class II epitope clusters in the N- and C-terminal regions of PfCSP 3D7 and an additional eight clusters through comparative analysis of 539 PfCSP sequence variants. A subset of these predicted class II epitope clusters was synthesized as peptides and assessed for HLA-DR binding in vitro. Further, we characterized the functional capacity of these peptides to prime and activate human peripheral blood mononuclear cells (PBMCs), by monitoring cytokine response profiles using MIMIC® technology (Modular IMmune In vitro Construct). Utilizing this decision framework, we found sufficient differential cellular activation and cytokine profiles among HLA-DR-matched PBMC donors to downselect class II epitope clusters for inclusion in a vaccine targeting PfCSP. Importantly, the downselected clusters are not highly conserved across PfCSP variants but rather, they overlap a hypervariable region (TH2R) in the C-terminus of the protein. We recommend assessing these class II epitope clusters within the context of a PfCSP vaccine, employing a test system capable of measuring immunogenicity across a broad set of HLA-DR alleles.

Identification, Selection and Immune Assessment of Liver Stage CD8 T Cell Epitopes From Plasmodium falciparum.

Immunization with radiation-attenuated sporozoites (RAS) has been shown to protect against malaria infection, primarily through CD8 T cell responses, but protection is limited based on parasite strain. Therefore, while CD8 T cells are an ideal effector population target for liver stage malaria vaccine development strategies, such strategies must incorporate conserved epitopes that cover a large range of class I human leukocyte antigen (HLA) supertypes to elicit cross-strain immunity across the target population. This approach requires identifying and characterizing a wide range of CD8 T cell epitopes for incorporation into a vaccine such that coverage across a large range of class I HLA alleles is attained. Accordingly, we devised an experimental framework to identify CD8 T cell epitopes from novel and minimally characterized antigens found at the pre-erythrocytic stage of parasite development. Through in silico analysis we selected conserved P. falciparum proteins, using P. vivax orthologues to establish stringent conservation parameters, predicted to have a high number of T cell epitopes across a set of six class I HLA alleles representative of major supertypes. Using the decision framework, five proteins were selected based on the density and number of predicted epitopes. Selected epitopes were synthesized as peptides and evaluated for binding to the class I HLA alleles in vitro to verify in silico binding predictions, and subsequently for stimulation of human T cells using the Modular IMmune In-vitro Construct (MIMIC®) technology to verify immunogenicity. By combining the in silico tools with the ex vivo high throughput MIMIC platform, we identified 15 novel CD8 T cell epitopes capable of stimulating an immune response in alleles across the class I HLA panel. We recommend these epitopes should be evaluated in appropriate in vivo humanized immune system models to determine their protective efficacy for potential inclusion in future vaccines.

Ecological correlates of species' roles in highly invaded seed dispersal networks.

Ecosystems with a mix of native and introduced species are increasing globally as extinction and introduction rates rise, resulting in novel species interactions. While species interactions are highly vulnerable to disturbance, little is known about the roles that introduced species play in novel interaction networks and what processes underlie such roles. Studying one of the most extreme cases of human-modified ecosystems, the island of O'ahu, Hawaii, we show that introduced species there shape the structure of seed dispersal networks to a greater extent than native species. Although both neutral and niche-based processes influenced network structure, niche-based processes played a larger role, despite theory predicting neutral processes to be predominantly important for islands. In fact, ecological correlates of species' roles (morphology, behavior, abundance) were largely similar to those in native-dominated networks. However, the most important ecological correlates varied with spatial scale and trophic level, highlighting the importance of examining these factors separately to unravel processes determining species contributions to network structure. Although introduced species integrate into interaction networks more deeply than previously thought, by examining the mechanistic basis of species' roles we can use traits to identify species that can be removed from (or added to) a system to improve crucial ecosystem functions, such as seed dispersal.

A potent novel vaccine adjuvant based on straight polyacrylate.

A structure-activity study was conducted to identify the structural characteristics underlying the adjuvant activity of straight (i.e. non-crosslinked) polyacrylate polymers (PAAs) in order to select a new PAA adjuvant candidate for future clinical development. The study revealed that the adjuvant effect of PAA was mainly influenced by polymer size (Mw) and dose. Maximal effects were obtained with large PAAs above 350 kDa and doses above 100 µg in mice. Small PAAs below 10 kDa had virtually no adjuvant effect. HPSEC analysis revealed that PAA polydispersity index and ramification had less impact on adjuvanticity. Heat stability studies indicated that residual persulfate could be detrimental to PAA stability. Hence, this impurity was systematically eliminated by diafiltration along with small Mw PAAs and residual acrylic acid that could potentially affect product safety, potency and stability. The selected PAA, termed SPA09, displayed an adjuvant effect that was superior to that of a standard emulsion adjuvant when tested with CMV-gB in mice, even in the absence of binding to the antigen. The induced immune response was dominated by strong IFNγ, IgG2c and virus neutralizing titers. The activity of SPA09 was then confirmed on human cells via the innate immune module of the human MIMIC® system.

Classifying Hawaiian plant species along a habitat generalist-specialist continuum: Implications for species conservation under climate change.

Plant communities on tropical high islands, such as the Hawaiian Islands, are predicted to experience rapid climate change, resulting in novel climates. If increased temperature and/or drought exceed plant species' current tolerances, species that are unable to adapt or shift ranges risk extinction. By definition, habitat generalists have a wide niche breadth and thrive in a variety of habitats, whereas habitat specialists have a narrow niche breadth, and typically thrive under more specific climatic characteristics (e.g., cold). The objectives of this study were to: (1) classify plant species in the Hawaiian Islands along a habitat generalist-specialist continuum; (2) independently test the validity of species rankings, using environmental and biogeographic ranges; and (3) identify species' life-history traits that predict species location along the continuum. We quantified specialization for 170 plant species using species co-occurrence data from over one thousand plots to rank species' realized habitat niche breadth using the Jaccard index. The distribution of species along this continuum differed by species biogeographic origin, with endemic plant species ranked on the specialist end and non-native plant species ranked on the generalist end. Habitat specialization rankings also differed for four of nine tested variables (while controlling for biogeographic origin): number of habitat moisture types, minimum elevation, number of Hawaiian Islands, and life form. Life form was the only trait tested that differed across the continuum, with woody species ranked as stronger generalists than herbaceous species; this pattern was particularly evident for non-native species. This indirect method of estimating species' potential climatic flexibility uses increasingly available large plant community data sets with output rankings which represent species' realized habitat niches. Identifying species and plant communities that are on the habitat specialist end of the continuum allows for their prioritization in conservation planning, as globally the loss of specialists is an indication of degradation.

Structure, spatial dynamics, and stability of novel seed dispersal mutualistic networks in Hawai'i.

Increasing rates of human-caused species invasions and extinctions may reshape communities and modify the structure, dynamics, and stability of species interactions. To investigate how such changes affect communities, we performed multiscale analyses of seed dispersal networks on O'ahu, Hawai'i. Networks consisted exclusively of novel interactions, were largely dominated by introduced species, and exhibited specialized and modular structure at local and regional scales, despite high interaction dissimilarity across communities. Furthermore, the structure and stability of the novel networks were similar to native-dominated communities worldwide. Our findings suggest that shared evolutionary history is not a necessary process for the emergence of complex network structure, and interaction patterns may be highly conserved, regardless of species identity and environment. Introduced species can quickly become well integrated into novel networks, making restoration of native ecosystems more challenging than previously thought.

Evaluation of the innate immunostimulatory potential of originator and non-originator copies of insulin glargine in an in vitro human immune model.

BACKGROUND: The manufacture of insulin analogs requires sophisticated production procedures which can lead to differences in the structure, purity, and/or other physiochemical properties of resultant products that can affect their biologic activity. Here, we sought to compare originator and non-originator copies of insulin glargine for innate immune activity and mechanisms leading to differences in these response profiles in an in vitro model of human immunity. METHODS: An endothelial/dendritic cell-based innate immune model was used to study antigen-presenting cell activation, cytokine secretion, and insulin receptor signalling pathways induced by originator and non-originator insulin glargine products. Mechanistic studies included signalling pathway blockade with specific inhibitors, analysis of the products in a Toll-like receptor reporter cell line assay, and natural insulin removal from the products by immunopurification. FINDINGS: All insulin glargine products elicited at least a minor innate immune response comparable to natural human insulin, but some lots of a non-originator copy product induced the elevated secretion of the cytokines, IL-8 and IL-6. In studies aimed at addressing the mechanisms leading to differential cytokine production by these products, we found (1) the inflammatory response was not mediated by bacterial contaminants, (2) the innate response was driven by the native insulin receptor through the MAPK pathway, and (3) the removal of insulin glargine significantly reduced their capacity to induce innate activity. No evidence of product aggregates was detected, though the presence of some high molecular weight proteins argues for the presence of insulin glargine dimers or others contaminants in these products. CONCLUSION: The data presented here suggests some non-originator insulin glargine product lots drive heightened in vitro human innate activity and provides preliminary evidence that changes in the biochemical composition of non-originator insulin glargine products (dimers, impurities) might be responsible for their greater immunostimulatory potential.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4+ T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4+ T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4+ T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4+ T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Phylogenetic classification of the world's tropical forests.

Knowledge about the biogeographic affinities of the world's tropical forests helps to better understand regional differences in forest structure, diversity, composition, and dynamics. Such understanding will enable anticipation of region-specific responses to global environmental change. Modern phylogenies, in combination with broad coverage of species inventory data, now allow for global biogeographic analyses that take species evolutionary distance into account. Here we present a classification of the world's tropical forests based on their phylogenetic similarity. We identify five principal floristic regions and their floristic relationships: (i) Indo-Pacific, (ii) Subtropical, (iii) African, (iv) American, and (v) Dry forests. Our results do not support the traditional neo- versus paleotropical forest division but instead separate the combined American and African forests from their Indo-Pacific counterparts. We also find indications for the existence of a global dry forest region, with representatives in America, Africa, Madagascar, and India. Additionally, a northern-hemisphere Subtropical forest region was identified with representatives in Asia and America, providing support for a link between Asian and American northern-hemisphere forests.

The in vitro MIMIC® platform reflects age-associated changes in immunological responses after influenza vaccination.

Increasing research and development costs coupled with growing concerns over healthcare expenditures necessitate the generation of pre-clinical testing models better able to predict the efficacy of vaccines, drugs and biologics. An ideal system for evaluating vaccine immunogenicity will not only be reliable but also physiologically relevant, able to be influenced by immunomodulatory characteristics such as age or previous exposure to pathogens. We have previously described a fully autologous human cell-based MIMIC® (Modular IMmune In vitro Construct) platform which enables the evaluation of innate and adaptive immunity in vitro, including naïve and recall responses. Here, we establish the ability of this module to display reduced antibody production and T cell activation upon in vitro influenza vaccination of cells from elderly adults. In the MIMIC® system, we observe a 2.7-4.2-fold reduction in strain-specific IgG production to seasonal trivalent influenza vaccine (TIV) in the elderly when compared to adults, as well as an age-dependent decline in the generation of functional antibodies. A parallel decline in IgG production with increasing age was detected via short-term ex vivo stimulation of B cells after in vivo TIV vaccination in the same cohort. Using MIMIC®, we also detect a reduction in the number but not proportion of TIV-specific multifunctional CD154+IFNγ+IL-2+TNFα+ CD4+ T cells in elderly adults. Inefficient induction of multifunctional helper T cells with TIV stimulation in MIMIC® despite a normalized number of initial CD4+ T cells suggests a possible mechanism for an impaired anti-TIV IgG response in elderly adults. The ability of the MIMIC® system to recapitulate differential age-associated responses in vitro provides a dynamic platform for the testing of vaccine candidates and vaccine enhancement strategies in a fully human model including the ability to interrogate specific populations, such as elderly adults.

Low redundancy in seed dispersal within an island frugivore community.

The low species diversity that often characterizes island ecosystems could result in low functional redundancy within communities. Flying foxes (large fruit bats) are important seed dispersers of large-seeded species, but their redundancy within island communities has never been explicitly tested. In a Pacific archipelago, we found that flying foxes were the sole effective disperser of 57 % of the plant species whose fruits they consume. They were essential for the dispersal of these species either because they handled >90 % of consumed fruit, or were the only animal depositing seeds away from the parent canopy, or both. Flying foxes were especially important for larger-seeded fruit (>13 mm wide), with 76 % of consumed species dependent on them for dispersal, compared with 31 % of small-seeded species. As flying foxes decrease in abundance, they cease to function as dispersers long before they become rare. We compared the seed dispersal effectiveness (measured as the proportion of diaspores dispersed beyond parent crowns) of all frugivores for four plant species in sites where flying foxes were, and were not, functionally extinct. At both low and high abundance, flying foxes consumed most available fruit of these species, but the proportion of handled diaspores dispersed away from parent crowns (quality) was significantly reduced at low abundance. Since alternative consumers (birds, rodents and land crabs) were unable to compensate as dispersers when flying foxes were functionally extinct, we conclude that there is almost no redundancy in the seed dispersal function of flying foxes in this island system, and potentially on other islands where they occur. Given that oceanic island communities are often simpler than continental communities, evaluating the extent of redundancy across different ecological functions on islands is extremely important.

Barriers to seed and seedling survival of once-common Hawaiian palms: the role of invasive rats and ungulates.

Mammalian herbivores can limit plant recruitment and affect forest composition. Loulu palms (Pritchardia spp.) once dominated many lowland ecosystems in Hawai'i, and non-native rats (Rattus spp.), ungulates (e.g. pigs Sus scrofa, goats Capra hircus) and humans have been proposed as major causes of their decline. In lowland wet forest, we experimentally determined the vulnerability of seeds and seedlings of two species of Pritchardia, P. maideniana and P. hillebrandii, by measuring their removal by introduced vertebrates; we also used motion-sensing cameras to identify the animals responsible for Pritchardia removal. We assessed potential seed dispersal of P. maideniana by spool-and-line tracking, and conducted captive-feeding trials with R. rattus and seeds and seedlings of both Pritchardia species. Seed removal from the forest floor occurred rapidly for both species: >50 % of Pritchardia seeds were removed from the vertebrate-accessible stations within 6 days and >80 % were removed within 22 days. Although rats and pigs were both common to the study area, motion-sensing cameras detected only rats (probably R. rattus) removing Pritchardia seeds from the forest floor. Captive-feeding trials and spool-and-line tracking revealed that vertebrate seed dispersal is rare; rats moved seeds up to 8 m upon collection and subsequently destroyed them (100 % mortality in 24-48 h in captivity). Surprisingly, seedlings did not suffer vertebrate damage in field trials, and although rats damaged seedlings in captivity, they rarely consumed them. Our findings are consistent with the hypothesis generated from palaeoecological studies, indicating that introduced rats may have assisted in the demise of native insular palm forests. These findings also imply that the seed stage of species in this Pacific genus is particularly vulnerable to rats; therefore, future conservation efforts involving Pritchardia should prioritize the reduction of rat predation on the plant recruitment stages preceding seedling establishment.

Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4). We compared the innate response and functional profiles of branded and US-generic enoxaparins from 2 manufacturers in either native or PF4-bound forms in an in vitro model of human immunity. In an analysis of 2 product lots from each manufacturer and multiple separate batches of protein-heparin complexes, branded enoxaparin was shown to be consistently nonstimulatory for innate responses, whereas US-generic enoxaparins generated variable immunostimulatory profiles depending on the enoxaparin lot used to prepare the PF4-LMWH complexes. Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins. Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study. Although these distinct biological and analytical profiles might be related to the composition and/or consistency of branded and US-generic enoxaparins included in our data set, further studies are warranted to elucidate the pathophysiological relevance of these in vitro findings.

Introduction to the Special Issue: Advances in island plant biology since Sherwin Carlquist's Island Biology.

Sherwin Carlquist's seminal publications-in particular his classic Island Biology, published in 1974-formulated hypotheses specific to island biology that remain valuable today. This special issue brings together some of the most interesting contributions presented at the First Island Biology Symposium hosted in Honolulu on 7-11 July 2014. We compiled a total of 18 contributions that present data from multiple archipelagos across the world and from different disciplines within the plant sciences. In this introductory paper, we first provide a short overview of Carlquist's life and work and then summarize the main findings of the collated papers. A first group of papers deals with issues to which Carlquist notably contributed: long-distance dispersal, adaptive radiation and plant reproductive biology. The findings of such studies demonstrate the extent to which the field has advanced thanks to (i) the increasing availability and richness of island data, covering many taxonomic groups and islands; (ii) new information from the geosciences, phylogenetics and palaeoecology, which allows us a more realistic understanding of the geological and biological development of islands and their biotas; and (iii) the new theoretical and methodological advances that allow us to assess patterns of abundance, diversity and distribution of island biota over large spatial scales. Most other papers in the issue cover a range of topics related to plant conservation on islands, such as causes and consequences of mutualistic disruptions (due to pollinator or disperser losses, introduction of alien predators, etc.). Island biologists are increasingly considering reintroducing ecologically important species to suitable habitats within their historic range and to neighbouring islands with depauperate communities of vertebrate seed dispersers, and an instructive example is given here. Finally, contributions on ecological networks demonstrate the usefulness of this methodological tool to advancing conservation management and better predicting the consequences of disturbances on species and interactions in the fragile insular ecosystems.

Island biology: looking towards the future.

Oceanic islands are renowned for the profound scientific insights that their fascinating biotas have provided to biologists during the past two centuries. Research presented at Island Biology 2014-an international conference, held in Honolulu, Hawaii (7-11 July 2014), which attracted 253 presenters and 430 participants from at least 35 countries(1)-demonstrated that islands are reclaiming a leading role in ecology and evolution, especially for synthetic studies at the intersections of macroecology, evolution, community ecology and applied ecology. New dynamics in island biology are stimulated by four major developments. We are experiencing the emergence of a truly global and comprehensive island research community incorporating previously neglected islands and taxa. Macroecology and big-data analyses yield a wealth of global-scale synthetic studies and detailed multi-island comparisons, while other modern research approaches such as genomics, phylogenetic and functional ecology, and palaeoecology, are also dispersing to islands. And, increasingly tight collaborations between basic research and conservation management make islands places where new conservation solutions for the twenty-first century are being tested. Islands are home to a disproportionate share of the world's rare (and extinct) species, and there is an urgent need to develop increasingly collaborative and innovative research to address their conservation requirements.