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The ongoing and often synergistic effects of habitat loss, invasive species, and climate change pose challenges for conservation and management as widespread species become greatly reduced, sometimes to a single small population. To address this problem, conservation biologists must consider using approaches like translocation to create new populations, reducing the probability of extinction by splitting a population into 2 or more populations in geographically distinct locales. The puaiohi Myadestes palmeri, an endangered Hawaiian forest bird, has a small population size (494; 95% CI: 414-580) and restricted range (~40 km2). One recovery plan objective involves translocating birds to higher elevation Hawaiian Islands. To evaluate translocation scenarios, we built upon previously developed population viability analysis models and considered how translocation regimes (initial harvest/population, number harvested/supplemented per event, harvest/supplementation interval, and length of harvest/supplementation) would affect both original and new populations. Furthermore, we modeled the puaiohi release population under 3 different conditions: a stable population, a predator-controlled environment, and a habitat improved in terms of resource availability. Our results indicated that while translocation offers hope of increasing puaiohi population size and decreasing extinction risk, success will depend on conditions at the release site. Furthermore, harvest and rearing of eggs to the juvenile stage or re-establishment of a captive breeding program may be necessary to provide enough birds to translocate, as the current wild population may not be productive enough to sustain levels of harvest necessary to successfully establish a new population.

We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule arrays (Simoa) with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (≤5000 vs. ∼500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency) from ∼5% to ∼50%. Bead read efficiency was increased by: a) improving the loading of beads into arrays of microwells by combining capillary and magnetic forces in a method called magnetic-meniscus sweeping (MMS); b) using a centrifugal washer to minimize bead loss during the assay; and, c) improved optics and image analysis to enable the analysis of more microwells. Using this approach, we developed an assay for IL-17A with a limit of detection (LOD) of 0.7 aM, 437-fold more sensitive than standard digital ELISA. A digital ELISA with improved sensitivity was used to measure IL-17A in 100 serum and plasma samples with 100% detectability, compared to 51% for standard digital ELISA. Low numbers of capture beads yielded improved LODs for IL-12p70 (0.092 aM), p24 (9.1 aM), and interferon alpha (45.9 aM). IL-4 and PSA showed no improvements in sensitivity using fewer beads, primarily due to low antibody loading on beads and increased non-specific binding, respectively. The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity.

We have developed an ultrasensitive multiplexed immunoassay using 384-well microtiter plates capable of detecting proteins at subfemtomolar concentrations that requires as little as 2.5 μL of sample. Arrays of up to 4 capture antibodies were patterned on the bottom of the wells of a 384-well plate either by directly printing the capture antibodies or by printing anti-peptide tag anchor antibodies and incubating these arrays with capture antibodies conjugated to the corresponding peptide tags ("customized" assays). Samples were incubated with the antibody arrays and shaken orbitally at 2000 rpm to achieve the greatest sensitivity. Chemiluminescence (CL) from immunocomplexes labeled with horseradish peroxidase was imaged across the entire plate to quantify the amount of protein bound to each antibody spot of the arrays. The 384-well assay had a throughput 5-fold greater than 96-well plates that was achieved from simultaneous imaging of CL in all 384-wells and the use of automated pipettors to allow parallel processing of 384 assays. We developed 4 assays based on the 384-well CL ELISA: a direct print assay for IL-10 (limit of detection (LOD) = 0.075 fM); a customized assay for IL-6 (0.22 fM); a customized pharmacokinetic (PK) assay for measuring adalimumab (7.3 pg/mL); and a customized 4-plex assay for IL-5 (0.1 fM), IL-6 (0.52 fM), IL-10 (0.2 fM), and TNF-α (3.2 fM). The sensitivity and precision of the cytokine assays were comparable to current ultrasensitive protein detection methods in 96-well formats. The PK assay for adalimumab was 650 times more sensitive than a commercially available 96-well plate ELISA. We used the 384-well CL ELISAs to measure endogenous levels of the cytokines in the serum and plasma of healthy humans: the mean concentrations and precision were comparable to those from 96-well immunoassays. This 384-well format with subfemtomolar sensitivity will enable ultrasensitive multiplexed immunoassays to be performed with higher throughput and lower sample volumes than currently possible, a particularly important capability for clinical studies in drug development.

Background Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A–negative, toxin B–positive (A−B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. Methods An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). Results There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23–17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). Conclusions We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

SummaryWith rapid urbanization worldwide, most people now live in cities, but the effects of urbanization on knowledge about the natural environment is not well studied. Due to the importance of Cymbidium to Chinese traditional culture, we tested how urbanization influences the distribution of orchid knowledge across various knowledge domains at risk of loss due to declining orchid populations. Participants in the Cymbidium trade were interviewed in three distinct urbanization-level jurisdictions in Sichuan, China: Puge (low urbanization), Huili (moderate urbanization) and Chengdu (high urbanization). Using photographic cue-cards of nine Cymbidium taxa, we assessed aggregate and specific knowledge held by 91 orchid collectors/traders across the urbanization gradient. Contrary to expectations, we found that urbanization and orchid knowledge were positively related, but this varied by knowledge type, with moderate urbanization showing significantly higher knowledge in two domains. Our findings suggest that a generalizable understanding of how urbanization affects knowledge must account for differences in knowledge types and geographic/cultural scales, with implications for biocultural diversity conservation in an increasingly urban world.

We have developed a customizable contact printed multiplex immunoassay capable of simultaneously measuring up to five analytes with attomolar sensitivities. This enzyme-linked immunosorbent assay (ELISA) was based on spotting different antibodies in a circular pattern at the bottom of a microtiter plate well. Unlike traditional antibody printing for ELISA that prints a capture antibody specific to a target of interest, in this ELISA we printed unique "anchor" antibodies at the well surface, each having a high affinity for a specific peptide target. By coupling each peptide to a unique assay capture antibody, this array of anchor antibodies enabled a customizable contact printed multiplex immunoassay workflow. As a proof of concept, we developed a 5-plex assay measuring interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 22 (IL-22), and tumor necrosis factor alpha (TNF-α). Measurements of these five analytes in serum and plasma correlated well between the method utilizing the anchor antibodies and peptides and the traditional capture antibody printing approach, with r2 values of 0.99, 0.93, 0.99, 0.96, and 0.75 for IL-5, IL-6, IL-10, IL-22, and TNFα, respectively. This approach makes customizable multiplex ultrasensitive ELISA available to laboratories without access to the precision printing instrumentation and will be useful for antibody screening, custom assay development, biomarker detection, and protein profiling for diagnostic applications.

This paper describes the need for technologies that improve analytical sensitivity to proteins to better define and monitor the progression from heath to disease over the course of an individual's life. These technologies have the potential to allow the early diagnosis of disease, and trigger treatments at the time when they have the greatest opportunity to be effective. We will describe a technology that we have developed for high sensitivity protein detection, namely, single molecule arrays (Simoa). Simoa is based on the capture of protein molecules on magnetic beads, labeling each protein with an enzyme, and counting of single enzyme labels on beads that are isolated in arrays of femtoliter wells. Simoa has enabled the detection of proteins at subfemtomolar concentrations in a variety of biological fluids. We describe the impact of higher sensitivity of proteins using Simoa on: less invasive testing; earlier detection of disease; providing biomarker baseline profiles for healthy individuals; testing of small sample volumes; monitoring of therapeutic efficacy; faster tests; and detection of proteins in complex samples. We also provide a perspective of how new technologies that allow the low-cost manufacture and miniaturization of Simoa could drive the next wave of analytical devices, including wearables.

Circulating microRNAs are biomarkers reported to be stable and translational across species. MicroRNA-122 (miR-122) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). We developed a single molecule, dynamic chemical labeling (DCL) assay to directly detect miR-122 in blood. The DCL assay specifically measured miR-122 directly from 10 μL of serum or plasma without any extraction steps, with a limit of detection of 1.32 pM that enabled the identification of DILI. Testing of 192 human serum samples showed that DCL accurately identified patients at risk of DILI after acetaminophen overdose (area under ROC curve 0.98 (95% CI; 0.96-1), P

Circulating microRNAs are biomarkers reported to be stable and translational across species. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free and amplification-free detection method for measuring miR-122 that has translational utility in context of DILI. We developed a single molecule dynamic chemical labelling (DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probe that contained a reactive amine instead of a nucleotide at a specific position in the sequence. The single molecule DCL assay specifically measured miR-122 directly from 10 µL of serum or plasma without any extraction steps, with a fit-for-purpose limit of detection of 1.32 pM. In 192 human serum samples, DCL accurately identified patients at risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P

We have characterized the sensitivity and kinetics of a multiplex immunoassay system based on detection of chemiluminescence (CL) at arrays of antibodies. This enzyme-linked immunosorbent assay (ELISA) was based on the spotting of different antibodies in a circular pattern at the bottom of a well of a microtiter plate. Sandwich immunocomplexes within each spot were labeled with horse radish peroxidase, and CL was generated locally to each spot in the array from turnover of luminol substrate. CL from the arrays across the plate was collected in single images; long exposure times were used to maximize sensitivity, and short exposure times were used to extend the dynamic range at higher signals. Image analysis was used to determine the intensity of light from each spot in the array, and intensity was converted to concentration of protein via comparison to a calibration curve. To determine the intrinsic sensitivity of the CL ELISA array, streptavidin horseradish peroxidase (SA-HRP) was captured on an array spotted with biotinylated detection antibodies. The limit of detection (LOD) of SA-HRP was 105 aM, or 3200 enzymes per 50 μL. A single-plex assay for prostate specific antigen (PSA) was developed that had an LOD of 79 aM when the microtiter plate was shaken orbitally, comparable to the most sensitive immunoassays reported to date. Normalization of CL signals in the PSA assay to signal per molecule of SA-HRP showed that the efficiency of the shaken assay was ~40%. When the plates were not shaken, the efficiency was ~4.5%, i.e., ~9-fold lower than when shaken. To better understand the theoretical basis of the sensitivity of these assays, we developed COMSOL numerical models of the binding kinetics at the array for plates that were shaken orbitally and those not shaken. Experimental data from the orbitally shaken PSA assay were best modeled by inertial mixing in a three-layer system that included a 8-μm-thick concentration boundary layer. Experimental data from the unshaken PSA assay were well modeled by diffusion-limited kinetics. A single-plex assay for IL-10 was developed with an LOD of 69 aM or 1.5 fg/mL, and used to measure this cytokine in plasma and serum of 10 healthy individuals. A 5-plex assay for IL-5, IL-6, IL-10, IL-22, and TNF-α was developed with LODs of 56 aM, 237 aM, 69 aM, 88 aM, and 373 aM, respectively. The assay was used to measure these 5 cytokines in the plasma and serum of the same individuals. The correlation in concentration of IL-10 measured in single-plex and multiplex assays was good (r2 = 0.89; bias = 14.5%). The factors that result in the high sensitivity of CL ELISA arrays—mostly high signal to noise ratio of extended chemiluminescent imaging—are discussed. This multiplex CL ELISA could be used for sensitive profiling of multiple proteins for in vitro diagnostics and biomarker detection in the development of therapeutics.

Featured Article: Rissin DM, Kan CW, Campbell TG, Stuart SC, Fournier DR, Song L, et al. Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations. Nat Biotechnol 2010;28:595–9.3 Proteins are the functional units of life. Berson et al. opened the field of protein measurements with the development of RIAs in the 1950s (1). In 1971, Engvall and Perlmann reported the ELISA (2) that has since been the staple for protein measurements. In 2010, when our article featured here was published, there were 325 proteins detectable in blood, of which 171 were Food and Drug Administration–approved tests of clinical value (3). Most of these proteins are measured using ELISA. When the human genome was sequenced, it was predicted that there should be nearly 4000 proteins in the human bloodstream (4). Where are the missing proteins? ELISA enabled detection down to picomolar concentrations, but this limit of quantification was insufficient to fully access the human blood proteome. The single-molecule array (Simoa) …

The Nightingale Reed-Warbler (Acrocephalus hiwae), a critically endangered songbird, is the last remaining of four reed-warbler species that once inhabited the Mariana Islands. The most recent population estimate for the species is 2915-3742 individuals distributed over the islands of Saipan (n = 2742, 95% CI = 1686-3956) and Alamagan (n = 946, 95% CI = 173-1000), which represents a significant decline for the species on Saipan. Though data on life history parameters such as survival rates are not widely available, other parameters such as clutch size and nest success have been studied. Given the extirpation of three of the four reed-warbler species from the Mariana Islands, the recent significant decline of the larger Saipan population, and the increasing threats faced by the species, it is critical to determine what additional life history information is needed to aid management decisions. Using Vortex, we developed stochastic population models to represent current reed-warbler population dynamics and used sensitivity analysis to identify the life history parameters to which the model was most sensitive. Baseline models without inbreeding indicated declines of the Nightingale Reed-Warbler populations on Saipan and Alamagan, as well as of the overall population. The inclusion of inbreeding further increases the modeled rate of population decline. Parameters such as initial population size, carrying capacity, and male survival have relatively little influence on reed-warbler population models, while female and juvenile survival and, to a lesser degree, fecundity measures, exhibit strong influences on the species' population dynamics. We recommend effort be placed on collecting updated survival data for all life stages, but particularly for females and juveniles. Given model predictions of vulnerability to extinction, we recommend management actions that may increase survival rates of juveniles and females.

The endangered Puaiohi (Myadestes palmeri) endemic to Kaua‘i is the island’s only remaining native thrush. Given its small population of ~500 birds, it is essential to understand conditions that affect the species’ recruitment and survival. Previous observations of Puaiohi suggested that weather may influence nest success and productivity, but no studies investigated this relationship empirically. Our goal was to investigate potential links between weather conditions (precipitation and temperature) in the Puaiohi’s range (~20 km2) and several measures of reproductive success, using published data from 1996–1998 and new data collected by the Kaua‘i Forest Bird Recovery Project from 2007–2009. Total rainfall in the previous wet season strongly and positively correlated with the majority of the nest success variables (three of four). Mean rainfall during the breeding season correlated positively with reproductive effort (attempts/season and length of breeding season) and with total reproductive outp...

Domestic cats (Felis catus) are a common household pet and also a notorious invasive species around the world. Because cat numbers have been increasing in many locations it is critical to work on management solutions that help to reduce threats posed by cats. With regard to cat behavior, one of the threats both to cats themselves and the species that they interact with is disease transmission. As part of a broader overview on applying cat behavior to management the focus of this review is to consider different types of cat behaviors and highlight how they relate to disease as a means to help inform management. Specifically, we focus on cat movement, foraging, and cat–human interactions as broad classes of cat behavior that can lead to acquisition and transmission of diseases. In addition, we review the diseases that are commonly harbored by cats, are of growing human health concern, and for which we have reasonable information. Finally, we review the main forms of cat management in order to provide a set of recommendations for use in addressing cat diseases.

Biological and cultural diversity are integrally linked, yet understanding how culture impacts biological extinction is limited. The orchid richness of Southwest China’s Sichuan Province is the second highest in China, but price speculation and overharvest have resulted in significant recent orchid population collapses. Due to the importance of Cymbidium orchids to Han Chinese traditional culture, this study sought to test how the decline of these populations related to four different orchid knowledge domains and various socio-demographic variables on the local scale. Interviews were conducted in eight villages in rural Liangshan Yi Autonomous Prefecture: four Han-majority and four Yi-majority. Using photographic-cue-cards of nine Cymbidium taxa, aggregate- and specific-knowledge were assessed of 15 randomly selected individuals per village (120 total), in three age groups. Results showed that species decline negatively impacted culturally important knowledge of orchids across all knowledge domains, regardless of pre-extinction orchid rarity, and regardless of socio-demographic variables. Following population collapse, the decline in perceived economic value of orchid knowledge outweighed the influence of cultural veneration that traditionally drove Cymbidium knowledge acquisition. These findings highlight how biodiversity loss not only negatively affects the ecology and environment, but also culture, with profound implications for cultural resilience and biocultural diversity conservation efforts.

The island of Kaua‘i, Hawai‘i has several remaining populations of endangered, endemic Hawaiian petrels (Pterodroma sandwichensis) and Newell's shearwaters (Puffinus newelli) that would be threatened by the presence of predatory mongooses (Herpestes javanicus). Despite over 200 putative sightings, 1 road-kill and 2 recent captures, it is not clear if mongooses have actually become established on Kaua‘i. Comparing road transects on three Hawaiian islands, we found mongooses present on O‘ahu and Maui where they are known to occur and no evidence of significant populations on Kaua‘i. Three population scenarios are presented that would account for the three specimens, the past sightings, and the absence of sightings on our transects on Kaua‘i.

We report a system and assay for performing fully-automated measurement of 6 proteins simultaneously with single molecule sensitivity. The system combines handling of samples, reagents, and consumables, with a module for imaging single molecule arrays (Simoa) to enable immunoassays that have high sensitivity (~fg/mL), are multiplexed, and are fully-automated. A 6-plex cytokine Simoa assay for IL-6, TNF-α, GM-CSF, IL-10, IL-1β, and IL-1α was developed on the system. The assays had limits of detection in the range 0.01-0.03pg/mL, and the average imprecision (CV) of the Simoa signal was 4.2%. This assay was used to measure the concentrations of these cytokines in the plasma of patients with Crohn's Disease (CD), before and after treatment with anti-TNF-α antibody drugs, and in the serum of Type 1 diabetics. Concentrations of TNF-α and IL-6 in the CD samples determined using the fully-automated, multiplex Simoa assay had good correlation with the manual, single-plex assays previously reported. Drug treatment caused reductions in the mean concentration of all 6 cytokines in the plasma of CD patients. The concentrations of 4 cytokines were significantly higher in diabetics compared to healthy controls. The system could enable the widespread, multiplexed measurement of protein biomarkers with low abundance.

We developed a simple immunoassay capable of differentially detecting toxin B from highly virulent strains of Clostridium difficile (BI/NAP-1/027) in stool. This assay can simultaneously confirm the presence of in vivo toxin production and provide strain-related information relevant to infection control epidemiology and disease prognosis.

How species come to be established on islands and their consequent adaptations and evolution are subjects that lie at the heart of much of ecology and evolutionary and conservation biology. On islands, small populations, limited gene diversity and flow, and simpler ecosystems facilitate our understanding of how species arrive and then adapt and evolve in new locations. One component of this is understanding how the transition from arrival to establishment may affect species and their parasites and diseases. Colonizing species may arrive without the full burdens of parasites weighing on their source populations, allowing them to reduce their energetic investments in immune defenses. However, reduced genetic diversity may also reduce populations through inbreeding depression or diminish capacity to evolve. Loss of genes and adaptation to novel environments may over time reduce the capacity of insular species to deal with new parasites or old ones that “catch up.” With increasing anthropogenic introduction of novel biotas, including parasites, to islands, the conservation of insular biodiversity becomes increasingly challenging, which in turn reduces our ability to study and understand both islands and diseases.

We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA) probe—containing a reactive amine instead of a nucleotide at a specific position in the sequence—for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase—containing an aldehyde group—that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)—an established biomarker of liver toxicity—extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use., Quanterix Corporation provided support in the form of salaries and research materials for authors [D.M.R. and D.C.D.]., DestiNA Genomics Ltd. provided support in the form of salaries and research materials for authors [B.L.-L., S.P., and H.I.].

The miconia (Miconia calvescens) invasion of the East Maui Watershed (EMW) started from a single introduction over 40 yr ago, establishing a nascent patch network spread across 20,000 ha. In 2012, an accelerated intervention strategy was implemented utilizing the Herbicide Ballistic Technology (HBT) platform in a Hughes 500D helicopter to reduce target densities of seven nascent patches in the EMW. In a 14-mo period, a total of 48 interventions eliminated 4,029 miconia targets, with an estimated 33% increase in operations and 168% increase in recorded targets relative to the adjusted means from 2005 to 2011 data (prior to HBT adoption). This sequence of interventions covered a total net area of 1,138 ha, creating a field mosaic of overlapping search coverage (saturation) for each patch (four to eight interventions per patch). Target density reduction for each patch fit exponential decay functions (R2 > 0.88, P < 0.05), with a majority of the target interventions spatially assigned to the highest saturation fields. The progressive decay in target density led to concomitant reductions in search efficiency (min ha−1) and herbicide use rate (grams ae ha−1) in subsequent interventions. Mean detection efficacy (± SE) between overlapping interventions (n = 41) was 0.62 ± 0.03, matching closely with the probability of detection for a random search operation and verifying imperfect (albeit precise) detection. The HBT platform increases the value of aerial surveillance operations with 98% efficacy in target elimination. Applying coverage saturation with an accelerated intervention schedule to known patch locations is an adaptive process for compensating imperfect detection and building intelligence with spatial and temporal relevance to the next operation.

Arctic terns Sterna paradisaea are noted for their extraordinary migration between Arctic and sub-Arctic breeding grounds and Antarctic wintering areas. Until recently, few data existed to document this migration, and none existed for North Pacific breeders. In this study, we tracked 6 Alaskan Arctic terns tagged with combined light geolocation and saltwater immersion tags through their fall migration. During fall 2007, these birds used several highly productive stopover locations to refuel during their southward migration: the California Current, the northern and southern Humboldt Current, and the Patagonian Shelf. At least 3 of the birds went on to win- ter in the Weddell Sea region of Antarctica, where Arctic terns from several Atlantic populations are also known to winter. Analysis of the first ever post-breeding behavioral data collected on this species showed that the birds foraged extensively in these staging areas, spending more time for- aging on days when they were located within staging areas during the fall migration. We also found that the birds were exclusively diurnal foragers, spending their nights standing out of the water and/or flying. Arctic terns likely face strict time constraints throughout the migration, timing stopovers to match production while simultaneously aiming to arrive at the wintering grounds with sufficient time remaining to complete the winter molt before returning north. Ecological dis- turbance at any of these locations could have serious consequences for many birds. Further, pre- dicted effects of climate change in the Weddell Sea region could have repercussions throughout the global Arctic tern population.

We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 μL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 μL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).

We have developed a highly sensitive immunoassay-called digital ELISA-that is based on the detection of single enzyme-linked immunocomplexes on beads that are sealed in arrays of femtoliter wells. Digital ELISA was designed to be highly efficient in the capturing of target proteins, labeling of these proteins, and their detection in single molecule arrays (SiMoA); in essence, the goal of the assay is to "capture every molecule, detect every molecule". Here we provide the theoretical basis for the design of this assay derived from simple equations based on bimolecular interactions. Using these equations and knowledge of the concentrations of reagents, the times of interactions, and the on- and off-rates of the molecular interactions for each step of the assay, it is possible to predict the number of immunocomplexes that are formed and detected by SiMoA. The unique ability of SiMoA to count single immunocomplexes and determine an average number of enzymes per bead (AEB), makes it possible to directly compare the number of molecules detected experimentally to those predicted by theory. These predictions compare favorably to experimental data generated for a digital ELISA for prostate specific antigen (PSA). The digital ELISA process is efficient across a range of antibody affinities (K(D)~10(-11) -10(-9) M), and antibodies with high on-rates (k(on)>10(5) M(-1) s(-1)) are predicted to perform best. The high efficiency of digital ELISA and sensitivity of SiMoA to enzyme label also makes it possible to reduce the concentration of labeling reagent, reduce backgrounds, and increasing the specificity of the approach. Strategies for dealing with the dissociation of antibody complexes over time that can affect the signals in an assay are also described.

This article reviews the biology, ecological effects, and management of the domestic cat (Felis catus) in the Pacific basin. The cat is one of the most controversial invasive species in the Pacific region because of its complex relations with humans. At one extreme, well-fed domestic house pets are allowed outdoors where they may hunt native animals; at the other, unsocialized feral cats have replaced native predators as apex predators or occupy a new niche on oceanic islands, where they have devastated native faunas. In the middle are stray cats that are still socialized around humans. Feral and stray cats can be reservoirs of diseases that infect free-roaming domestic cats, humans, and wildlife. Given these problems, the best response would be to keep domestic cats indoors, restrict cat breeding, and remove feral populations. However, most Pacific basin societies have failed to reach a consensus on the cat problem, so solutions are ad hoc, often lacking in any scientific basis, and reflect our conflicting views. Compromise management might best fall into three broad classes: (1) eradication of cats should be confined to islands and other areas of high native biodiversity where reintroduction can be prevented; (2) in a landscape of low or moderate biological value, efforts should be made to educate the public to reduce the impact of their cats on remaining wildlife, while excluding cats from “islands” of elevated biodiversity values or human sensitivity; (3) in drastically simplified urban ecosystems, management perhaps should occur only in response to local complaints.

We developed an epidemiological model of avian malaria (Plasmodium relictum) across an altitudinal gradient on the island of Hawaii that includes the dynamics of the host, vector, and parasite. This introduced mosquito-borne disease is hypothesized to have contributed to extinctions and major shifts in the altitudinal distribution of highly susceptible native forest birds. Our goal was to better understand how biotic and abiotic factors influence the intensity of malaria transmission and impact on susceptible populations of native Hawaiian forest birds. Our model illustrates key patterns in the malaria-forest bird system: high malaria transmission in low-elevation forests with minor seasonal or annual variation in infection; episodic transmission in mid-elevation forests with site-to-site, seasonal, and annual variation depending on mosquito dynamics; and disease refugia in high-elevation forests with only slight risk of infection during summer. These infection patterns are driven by temperature and rainfall effects on parasite incubation period and mosquito dynamics across an elevational gradient and the availability of larval habitat, especially in mid-elevation forests. The results from our model suggest that disease is likely a key factor in causing population decline or restricting the distribution of many susceptible Hawaiian species and preventing the recovery of other vulnerable species. The model also provides a framework for the evaluation of factors influencing disease transmission and alternative disease control programs, and to evaluate the impact of climate change on disease cycles and bird populations.

BACKGROUNDMeasurement of prostate-specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been hindered by the limit of quantification of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, postsurgical PSA is typically undetectable with current assay methods. Evidence suggests, however, that more sensitive determination of PSA status following RP could improve assessment of patient prognosis and response to treatment and better target secondary therapy for those who may benefit most. We developed an investigational digital immunoassay with a limit of quantification 2 logs lower than current ultrasensitive third-generation PSA assays.METHODSWe developed reagents for a bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. We characterized analytical performance, compared its accuracy with a commercially available test, and analyzed longitudinal serum samples from a pilot study of 33 RP patients.RESULTSThe assay exhibited a functional sensitivity (20% interassay CV) CONCLUSIONSThe robust 2-log improvement in limit of quantification relative to current ultrasensitive assays and the validated analytical performance of the assay allow for accurate assessment of PSA status after RP.

The quantitative measurement of inflammatory cytokines in blood has been limited by insufficient sensitivity of conventional immunoassays. This limitation has prevented the widespread clinical monitoring of cytokine concentrations in chronic inflammatory diseases. We applied a sensitive, single molecule detection technology to measure TNF-α and IL-6 in the plasma of patients with Crohn's disease (CD), before and after treatment with anti-TNF-α therapy. Plasma from 17 patients with CD was collected prior to initiation of anti-TNF-α therapy, and the Crohn's disease activity index (CDAI) was determined for each patient. A sub-set of these patients returned for follow up 12 weeks after treatment started. Plasma from age- and gender-matched controls was also collected. Digital ELISAs were developed for TNF-α and IL-6, and the plasma concentrations of these cytokines were determined using digital ELISA. The limits of detection of the TNF-α and IL-6 digital ELISAs were 0.008 pg/mL and 0.006 pg/mL, respectively. Both cytokines were detected in all samples using digital ELISA and the concentrations of TNF-α and IL-6 in the plasma of patients with CD were (3.6 ± 0.9) pg/mL and (10.9 ± 11.2) pg/mL, respectively. TNF-α levels in patients and healthy controls were not significantly different, but the IL-6 levels in plasma were significantly elevated in patients compared to controls. After therapy, the mean reduction of the concentrations of free TNF-α and IL-6 were 46% and 58%, respectively. Digital ELISA provided the first quantitative measurements of TNF-α and IL-6 concentrations in the plasma of all patients in a population with CD. The changes in cytokine concentrations after therapy—which could be quantified because of the high sensitivity of digital ELISA—could be used for monitoring therapeutic efficacy.

We report a method for combining the detection of single molecules (digital) and an ensemble of molecules (analog) that is capable of detecting enzyme label from 10(-19) M to 10(-13) M, for use in high sensitivity enzyme-linked immunosorbent assays (ELISA). The approach works by capturing proteins on microscopic beads, labeling the proteins with enzymes using a conventional multistep immunosandwich approach, isolating the beads in an array of 50-femtoliter wells (Single Molecule Array, SiMoA), and detecting bead-associated enzymatic activity using fluorescence imaging. At low concentrations of proteins, when the ratio of enzyme labels to beads is less than ∼1.2, beads carry either zero or low numbers of enzymes, and protein concentration is quantified by counting the presence of "on" or "off" beads (digital regime). (1) At higher protein concentrations, each bead typically carries multiple enzyme labels, and the average number of enzyme labels present on each bead is quantified from a measure of the average fluorescence intensity (analog regime). Both the digital and analog concentration ranges are quantified by a common unit, namely, average number of enzyme labels per bead (AEB). By combining digital and analog detection of singulated beads, a linear dynamic range of over 6 orders of magnitude to enzyme label was achieved. Using this approach, an immunoassay for prostate specific antigen (PSA) was developed. The combined digital and analog PSA assay provided linear response over approximately four logs of concentration ([PSA] from 8 fg/mL to 100 pg/mL or 250 aM to 3.3 pM). This approach extends the dynamic range of ELISA from picomolar levels down to subfemtomolar levels in a single measurement.

The ability to detect single protein molecules in blood could accelerate the discovery and use of more sensitive diagnostic biomarkers. To detect low-abundance proteins in blood, we captured them on microscopic beads decorated with specific antibodies and then labeled the immunocomplexes (one or zero labeled target protein molecules per bead) with an enzymatic reporter capable of generating a fluorescent product. After isolating the beads in 50-fl reaction chambers designed to hold only a single bead, we used fluorescence imaging to detect single protein molecules. Our single-molecule enzyme-linked immunosorbent assay (digital ELISA) approach detected as few as approximately 10-20 enzyme-labeled complexes in 100 microl of sample (approximately 10(-19) M) and routinely allowed detection of clinically relevant proteins in serum at concentrations (

Summary We outline a functional management model for the eradication of incipient populations of invasive species that avoids reliance on official governmental response. This model involves formation of informal multi-partner committees that utilise outside funding to achieve pest-management goals. We describe why such a system was needed in Hawaii, how it is structured, how it operates, its achievements, and its advantages and limitations. Fragmented and incomplete governmental authorities are currently the rule for invasive-species management in many parts of the world and typically lead to non-response or an ineffective response. The model we describe serves the useful function of allowing eradication of incipient pests to proceed while comprehensive biosecurity programs are devised through more traditional governmental channels.

Many species of tropical seabirds rely on subsurface predators such as tuna and dol- phins to drive prey close to the ocean's surface. We observed seabird foraging events from fishing vessels around the island of Oahu, Hawaii, to determine the prevalence and relative importance of different subsurface predators to seabird foraging. Sixty-nine seabird foraging events were observed, in 62 of which the presence and/or identity of subsurface predators could be determined. Of those 62, all but two involved subsurface predators and accounted for 99.8% of all foraging birds. Skipjack tuna Katsuwonus pelamis were the predominant (81%) subsurface predator involved in seabird feed- ing events, with odontocetes Stenella attenuata and Pseudorca crassidens (8%), mahimahi Coryphaena hippurus (6%), and yellowfin tuna Thunnus albacares (2%) being less common. Wedge- tailed shearwaters Puffinus pacificus and brown noddies Anous stolidus foraged in association with tuna schools more often than would be expected by chance alone. Red-footed boobies Sula sula, in contrast, did not associate with any subsurface predator in greater proportion than what would be expected by chance. Nine additional seabird species were observed foraging in association with sub- surface predators, but not in numbers large enough for statistical analysis. This study highlights the value of regional investigations able to uncover the variable nature of seabird-subsurface-predator associations. The finding that, in Hawaii, at least 2 species of seabirds showed tight dependence on tuna underlines the need for tuna fishery management to address the potential indirect ecological effects of fishing.

Disease detection at the molecular level is driving the emerging revolution of early diagnosis and treatment. A challenge facing the field is that protein biomarkers for early diagnosis can be present in very low abundance. The lower limit of detection with conventional immunoassay technology is the upper femtomolar range (10(-13) M). Digital immunoassay technology has improved detection sensitivity three logs, to the attomolar range (10(-16) M). This capability has the potential to open new advances in diagnostics and therapeutics, but such technologies have been relegated to manual procedures that are not well suited for efficient routine use. We describe a new laboratory instrument that provides full automation of single-molecule array (Simoa) technology for digital immunoassays. The instrument is capable of single-molecule sensitivity and multiplexing with short turnaround times and a throughput of 66 samples/h. Singleplex and multiplexed digital immunoassays were developed for 16 proteins of interest in cardiovascular, cancer, infectious disease, neurology, and inflammation research. The average sensitivity improvement of the Simoa immunoassays versus conventional ELISA was1200-fold, with coefficients of variation of10%. The potential of digital immunoassays to advance human diagnostics was illustrated in two clinical areas: traumatic brain injury and early detection of infectious disease.

Quanterix and Stratec Biomedical have developed an automated analyzer that enables the measurement of subfemtomolar concentrations of proteins. The development of this technology focusing on the custom optical module and microfluidic disc is outlined below.

Synopsis This study uses a spatially explicit microclimate/biophysical approach to examine the potential distribution of the Po’ouli on Maui to find either new habitats to search for existence or refine search efforts in previously occupied areas. We used specific physiological and behavioral ecology bird data, and Po’ouli morphological and spectral data obtained from museum specimens to address ecological and conservation-related questions about the Po’ouli that are otherwise very difficult to quantify. Laboratory and field tested microclimate and biophysical—behavioral animal computer models were integrated with remote sensing technologies. To show that the generic microclimate and endotherm models can predict metabolic and water loss requirements of Hawaiian Honeycreepers, we used the 2 species with known physiological properties, the Hawaiian Amakihi, Hemignathus virens, and the Hawaiian Anianiau, Hemignathus parvus. Predictions were within experimental measurement error of the laboratory measurements. Then using field rather than laboratory conditions as input data, we predict the field distribution of the Amakihi on Maui as the first spatial test of the models applied to birds. Results are consistent with Amakihi field distribution data. Fossils show that the Po’ouli once lived on Maui at low elevations in dry/mesic habitats on a likely diet of native tree snails and insects. The arrival of lethal mosquito-borne avian malaria in Hawaii exterminated low elevation Po’ouli forcing a population shift to mountain rainforests and possibly a snail diet instead of insects. To explore the maximum consequences of such a diet shift we assumed exclusive diets of snails versus insects at both low and high elevations. Snail diets require � 4 times higher foraging rates than do insect diets, making a predominantly snail diet an unlikely prospect for the Po’ouli. Landscape scale simulations suggest that a snail diet would force a Po’ouli distribution inconsistent with observations. A predominantly insect diet is consistent with distribution observations. We show that as local environmental conditions change across the landscape in space and diurnal/seasonal time it is possible to quantify animal physiological and behavioral consequences of those variations in their local environment. This enables quantification of the requisite spatial and temporal distribution and amount or availability of resources that may affect species’ potential for survival, growth, reproduction and distribution.

The past quarter century has seen an unprecedented increase in the number of new and emerging infectious diseases throughout the world, with serious implications for human and wildlife populations. We examined host persistence in the face of introduced vector-borne diseases in Hawaii, where introduced avian malaria and introduced vectors have had a negative impact on most populations of Hawaiian forest birds for nearly a century. We studied birds, parasites, and vectors in nine study areas from 0 to 1,800 m on Mauna Loa Volcano, Hawaii from January to October, 2002. Contrary to predictions of prior work, we found that Hawaii amakihi (Hemignathus virens), a native species susceptible to malaria, comprised from 24.5% to 51.9% of the avian community at three low-elevation forests (55–270 m). Amakihi were more abundant at low elevations than at disease-free high elevations, and were resident and breeding there. Infection rates were 24–40% by microscopy and 55–83% by serology, with most infected individuals experiencing low-intensity, chronic infections. Mosquito trapping and diagnostics provided strong evidence for year-round local transmission. Moreover, we present evidence that Hawaii amakihi have increased in low elevation habitats on southeastern Hawaii Island over the past decade. The recent emergent phenomenon of recovering amakihi populations at low elevations, despite extremely high prevalence of avian malaria, suggests that ecological or evolutionary processes acting on hosts or parasites have allowed this species to recolonize low-elevation habitats. A better understanding of the mechanisms allowing coexistence of hosts and parasites may ultimately lead to tools for mitigating disease impacts on wildlife and human populations.

Quanterix and Stratec Biomedical have developed an instrument that enables the automated measurement of multiple proteins at concentration ~1000 times lower than existing immunoassays. The instrument is based on Quanterix’s proprietary Single Molecule Array technology (Simoa™ ) that facilitates the detection and quantification of biomarkers previously difficult to measure, thus opening up new applications in life science research and in-vitro diagnostics. Simoa is based on trapping individual beads in arrays of femtoliter-sized wells that, when imaged with sufficient resolution, allows for counting of single molecules associated with each bead. When used to capture and detect proteins, this approach is known as digital ELISA (Enzyme-linked immunosorbent assay). The platform developed is a merger of many science and engineering disciplines. This paper concentrates on the optical technologies that have enabled the development of a fully-automated single molecule analyzer. At the core of the system is a custom, wide field-of-view, fluorescence microscope that images arrays of microwells containing single molecules bound to magnetic beads. A consumable disc containing 24 microstructure arrays was developed previously in collaboration with Sony DADC. The system cadence requirements, array dimensions, and requirement to detect single molecules presented significant optical challenges. Specifically, the wide field-of-view needed to image the entire array resulted in the need for a custom objective lens. Additionally, cost considerations for the system required a custom solution that leveraged the image processing capabilities. This paper will discuss the design considerations and resultant optical architecture that has enabled the development of an automated digital ELISA platform.

This paper describes the self-assembly of small objects--polyhedral metal plates with largest dimensions of 10 to 30 microm--into highly ordered, three-dimensional arrays. The plates were fabricated using photolithography and electrodeposition techniques, and the faces of the plates were functionalized to be hydrophobic or hydrophilic using self-assembled monolayers (SAMs). Self-assembly occurs in water through capillary interactions between thin films of a hydrophobic liquid (a liquid prepolymer adhesive) coated onto the hydrophobic faces of the plates; coalescence of the adhesive films reduces the interfacial free energy of the system and drives self-assembly. By altering the size and surface-patterning of the plates, the external morphologies of the aggregates were varied. Curing the adhesive furnished mechanically stable aggregates that were characterized by scanning electron microscopy (SEM). For assemblies formed by plates partially composed of a sacrificial material, a subsequent etching step furnished fully open, three-dimensional microstructures. This work validates the use of capillary interactions for three-dimensional mesoscale self-assembly in the 10-microm-size regime and opens new avenues for the fabrication of complex, three-dimensional microscructures.

Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.

This paper describes the fabrication and operating principles of a reconfigurable diffraction grating based on a microfluidic device. The device consists of an array of microscopic channels (50 μm wide and 20 μm deep) defined by the conformal contact between a transparent elastomeric material that has an embossed surface relief and a flat glass substrate. The microchannels can be filled reversibly with fluids (gas, aqueous solutions, or organic solutions). The difference in index of refraction between the fluid in the array of microchannels and the structural elastomeric solid generates a difference in the phase of the light passing through the device; absorption by the fluid changes the amplitude of the light. Both of these effects give rise to diffraction. The gratings are reconfigured by pumping liquids with different indices of refraction and optical densities through the microchannels. In these experiments, the devices exhibited maximum depths of modulation of ∼20 dB and switching times of ∼50 ms; the volume of liquid sampled by the incident light was about 8 nl. The potential application of these devices as sensing elements in micro total analysis systems (μTAS) and as actuators is evaluated.

This paper describes a microfluidic system in which fluids are pumped by centrifugal force through microscopic channels defined in a plastic disk in order to perform complex analytical processes. The channels are created either by casting poly(dimethylsiloxane) against molds fabricated by photolithography or by conventional machining of poly(methyl methyacrylate). The channels have a wide range of diameters (5 μm−0.5 mm) and depths (16 μm−3 mm). Fluids are loaded into reservoirs near the center of the disk, the disk is rotated on the shaft of a simple motor at 60−3000 rpm, and the fluids are pumped outward by centrifugal force through microfluidic networks. The control of flow in the time domain, i.e., gating, is achieved by the use of passive valves based on capillary forces. Flow rates ranging from 5 nL/s to >0.1 mL/s have been achieved using channels of different dimensions and different rates of rotation. The method of pumping is insensitive to many physicochemical properties of the liquid, such as pH...

This paper describes a procedure for rapidly and conveniently prototyping microfluidic devices that are useful with aqueous solutions. A design (with diameters of channels 20 µm) is created in a computer-aided design program and printed at high resolution on a transparency. This transparency is used as a mask in photolithography to create a master in positive relief photoresist: casting poly(dimethyl siloxane) (PDMS) against this master yields a polymeric replica containing a network of bas-relief channels. The channels are closed and sealed irreversibly by oxidizing the replica and another flat substrate (PDMS, glass, silicon, silicon oxide) in an oxygen plasma and bringing the two surfaces into conformal contact. Oxidation of the polymer allows the formation of a seal without using adhesives; it also generates channels that support electro-osmotic flow (EOF) and fill easily with aqueous solutions. Two microfluidic devices - a fluidic switch and a side channel flow controller - have been fabricated using this rapid prototyping methodology. These devices were tested using aqueous solutions as the test fluid and actuated by EOF.