Polymerization-based signal amplification for paper-based immunoassays

Diagnostic tests in resource-limited settings require technologies that are affordable and easy to use with minimal infrastructure. Colorimetric detection methods that produce results that are readable by eye, without reliance on specialized and expensive equipment, have great utility in these settings. We report a colorimetric method that integrates a paper-based immunoassay with a rapid, visible-light-induced polymerization to provide high visual contrast between a positive and a negative result. Using Plasmodium falciparum histidine-rich protein 2 as an example, we demonstrate that this method allows visual detection of proteins in complex matrices such as human serum and provides quantitative information regarding analyte levels when combined with cellphone-based imaging. It also allows the user to decouple the capture of analyte from signal amplification and visualization steps.Bill & Melinda Gates Foundation (Award 51308)United States. Defense Advanced Research Projects Agency (HR0011-12-2-0010)National Science Foundation (U.S.). Graduate Research FellowshipBurroughs Wellcome Fund (Career Award at the Scientific Interface

Enhanced responses of the anterior cingulate cortex neurones to colonic distension in viscerally hypersensitive rats

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65998/1/jphysiol.2005.096073.pd

Diversity, host specialization, and geographic structure of filarial nematodes infecting Malagasy bats

We investigated filarial infection in Malagasy bats to gain insights into the diversity of these parasites and explore the factors shaping their distribution. Samples were obtained from 947 individual bats collected from 52 sites on Madagascar and representing 31 of the 44 species currently recognized on the island. Samples were screened for the presence of micro-and macro-parasites through both molecular and morphological approaches. Phylogenetic analyses showed that filarial diversity in Malagasy bats formed three main groups, the most common represented by Litomosa spp. infecting Miniopterus spp. (Miniopteridae); a second group infecting Pipistrellus cf. hesperidus (Vespertilionidae) embedded within the Litomosoides cluster, which is recognized herein for the first time from Madagascar; and a third group composed of lineages with no clear genetic relationship to both previously described filarial nematodes and found in M. griveaudi, Myotis goudoti, Neoromicia matroka (Vespertilionidae), Otomops madagascariensis (Molossidae), and Paratriaenops furculus (Hipposideridae). We further analyzed the infection rates and distribution pattern of Litomosa spp., which was the most diverse and prevalent filarial taxon in our sample. Filarial infection was disproportionally more common in males than females in Miniopterus spp., which might be explained by some aspect of roosting behavior of these cave-dwelling bats. We also found marked geographic structure in the three Litomosa clades, mainly linked to bioclimatic conditions rather than host-parasite associations. While this study demonstrates distinct patterns of filarial nematode infection in Malagasy bats and highlights potential drivers of associated geographic distributions, future work should focus on their alpha taxonomy and characterize arthropod vectors

Enhancing sampling design in mist-net bat surveys by accounting for sample size optimization

The advantages of mist-netting, the main technique used in Neotropical bat community studies to date, include logistical implementation, standardization and sampling representativeness. Nonetheless, study designs still have to deal with issues of detectability related to how different species behave and use the environment. Yet there is considerable sampling heterogeneity across available studies in the literature. Here, we approach the problem of sample size optimization. We evaluated the common sense hypothesis that the first six hours comprise the period of peak night activity for several species, thereby resulting in a representative sample for the whole night. To this end, we combined re-sampling techniques, species accumulation curves, threshold analysis, and community concordance of species compositional data, and applied them to datasets of three different Neotropical biomes (Amazonia, Atlantic Forest and Cerrado). We show that the strategy of restricting sampling to only six hours of the night frequently results in incomplete sampling representation of the entire bat community investigated. From a quantitative standpoint, results corroborated the existence of a major Sample Area effect in all datasets, although for the Amazonia dataset the six-hour strategy was significantly less species-rich after extrapolation, and for the Cerrado dataset it was more efficient. From the qualitative standpoint, however, results demonstrated that, for all three datasets, the identity of species that are effectively sampled will be inherently impacted by choices of sub-sampling schedule. We also propose an alternative six-hour sampling strategy (at the beginning and the end of a sample night) which performed better when resampling Amazonian and Atlantic Forest datasets on bat assemblages. Given the observed magnitude of our results, we propose that sample representativeness has to be carefully weighed against study objectives, and recommend that the trade-off between logistical constraints and additional sampling performance should be carefully evaluated

Met-Independent Hepatocyte Growth Factor-mediated regulation of cell adhesion in human prostate cancer cells

BACKGROUND: Prostate cancer cells communicate reciprocally with the stromal cells surrounding them, inside the prostate, and after metastasis, within the bone. Each tissue secretes factors for interpretation by the other. One stromally-derived factor, Hepatocyte Growth Factor (HGF), was found twenty years ago to regulate invasion and growth of carcinoma cells. Working with the LNCaP prostate cancer progression model, we found that these cells could respond to HGF stimulation, even in the absence of Met, the only known HGF receptor. The new HGF binding partner we find on the cell surface may help to clarify conflicts in the past literature about Met expression and HGF response in cancer cells. METHODS: We searched for Met or any HGF binding partner on the cells of the PC3 and LNCaP prostate cancer cell models, using HGF immobilized on agarose beads. By using mass spectrometry analyses and sequencing we have identified nucleolin protein as a novel HGF binding partner. Antibodies against nucleolin (or HGF) were able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. Western blots, RT-PCR, and immunohistochemistry were used to assess nucleolin levels during prostate cancer progression in both LNCaP and PC3 models. RESULTS: We have identified HGF as a major signaling component of prostate stromal-conditioned media (SCM) and have implicated the protein nucleolin in HGF signal reception by the LNCaP model prostate cancer cells. Antibodies that silence either HGF (in SCM) or nucleolin (on the cell surfaces) eliminate the adhesion-stimulatory effects of the SCM. Likewise, addition of purified HGF to control media mimics the action of SCM. C4-2, an LNCaP lineage-derived, androgen-independent human prostate cancer cell line, responds to HGF in a concentration-dependent manner by increasing its adhesion and reducing its migration on laminin substratum. These HGF effects are not due to shifts in the expression levels of laminin-binding integrins, nor can they be linked to expression of the known HGF receptor Met, as neither LNCaP nor clonally-derived C4-2 sub-line contain any detectable Met protein. Even in the absence of Met, small GTPases are activated, linking HGF stimulation to membrane protrusion and integrin activation. Membrane-localized nucelolin levels increase during cancer progression, as modeled by both the PC3 and LNCaP prostate cancer progression cell lines. CONCLUSION: We propose that cell surface localized nucleolin protein may function in these cells as a novel HGF receptor. Membrane localized nucleolin binds heparin-bound growth factors (including HGF) and appears upregulated during prostate cancer progression. Antibodies against nucleolin are able to ameliorate the stimulatory effects of HGF on met-negative prostate cancer cells. HGF-nucleolin interactions could be partially responsible for the complexity of HGF responses and met expression reported in the literature

The Evolution of the Major Hepatitis C Genotypes Correlates with Clinical Response to Interferon Therapy

Patients chronically infected with hepatitis C virus (HCV) require significantly different durations of therapy and achieve substantially different sustained virologic response rates to interferon-based therapies, depending on the HCV genotype with which they are infected. There currently exists no systematic framework that explains these genotype-specific response rates. Since humans are the only known natural hosts for HCV-a virus that is at least hundreds of years old-one possibility is that over the time frame of this relationship, HCV accumulated adaptive mutations that confer increasing resistance to the human immune system. Given that interferon therapy functions by triggering an immune response, we hypothesized that clinical response rates are a reflection of viral evolutionary adaptations to the immune system.We have performed the first phylogenetic analysis to include all available full-length HCV genomic sequences (n = 345). This resulted in a new cladogram of HCV. This tree establishes for the first time the relative evolutionary ages of the major HCV genotypes. The outcome data from prospective clinical trials that studied interferon and ribavirin therapy was then mapped onto this new tree. This mapping revealed a correlation between genotype-specific responses to therapy and respective genotype age. This correlation allows us to predict that genotypes 5 and 6, for which there currently are no published prospective trials, will likely have intermediate response rates, similar to genotype 3. Ancestral protein sequence reconstruction was also performed, which identified the HCV proteins E2 and NS5A as potential determinants of genotype-specific clinical outcome. Biochemical studies have independently identified these same two proteins as having genotype-specific abilities to inhibit the innate immune factor double-stranded RNA-dependent protein kinase (PKR).An evolutionary analysis of all available HCV genomes supports the hypothesis that immune selection was a significant driving force in the divergence of the major HCV genotypes and that viral factors that acquired the ability to inhibit the immune response may play a role in determining genotype-specific response rates to interferon therapy

A review of methods for assessment of the rate of gastric emptying in the dog and cat: 1898-2002

Gastric emptying is the process by which food is delivered to the small intestine at a rate and in a form that optimizes intestinal absorption of nutrients. The rate of gastric emptying is subject to alteration by physiological, pharmacological, and pathological conditions. Gastric emptying of solids is of greater clinical significance because disordered gastric emptying rarely is detectable in the liquid phase. Imaging techniques have the disadvantage of requiring restraint of the animal and access to expensive equipment. Radiographic methods require administration of test meals that are not similar to food. Scintigraphy is the gold standard method for assessment of gastric emptying but requires administration of a radioisotope. Magnetic resonance imaging has not yet been applied for assessment of gastric emptying in small animals. Ultrasonography is a potentially useful, but subjective, method for assessment of gastric emptying in dogs. Gastric tracer methods require insertion of gastric or intestinal cannulae and are rarely applied outside of the research laboratory. The paracetamol absorption test has been applied for assessment of liquid phase gastric emptying in the dog, but requires IV cannulation. The gastric emptying breath test is a noninvasive method for assessment of gastric emptying that has been applied in dogs and cats. This method can be carried out away from the veterinary hospital, but the effects of physiological and pathological abnormalities on the test are not known. Advances in technology will facilitate the development of reliable methods for assessment of gastric emptying in small animals

Paper-based diagnostics in the antigen-depletion regime: High-density immobilization of rcSso7d-cellulose-binding domain fusion proteins for efficient target capture

© 2017 Elsevier B.V. In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760 μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system

Design Principles for Enhancing Sensitivity in Paper-Based Diagnostics via Large-Volume Processing

In this work, we characterize the impact of large-volume processing upon the analytical sensitivity of flow-through paper-based immunoassays. Larger sample volumes feature greater molar quantities of available analyte, but the assay design principles which would enable the rapid collection of this dilute target are ill-defined. We developed a finite-element model to explore the operating conditions under which processing large sample volumes via pressure-driven convective flow would yield an improved binding signal. Our simulation results underscore the importance of establishing a high local concentration of the analyte-binding species within the porous substrate. This elevated abundance serves to enhance the binding kinetics, matching the time scale of target capture to the period during which the sample is in contact with the test zone (i.e., the effective residence time). These findings were experimentally validated using the rcSso7d-cellulose-binding domain (CBD) fusion construct, a bifunctional binding protein which adsorbs to cellulose in high abundance. As predicted by our modeling efforts, the local concentration achieved using the rcSso7d-CBD species is uniquely enabling for sensitivity enhancement through large-volume processing. The rapid analyte depletion which occurs at this high surface density also permits the processing of large sample volumes within practical time scales and flow regimes. Using these findings, we present guidance for the optimal means of processing large sample volumes for enhanced assay sensitivity