Llama Single Domain Antibodies Specific for the 7 Botulinum Neurotoxin Serotypes as Heptaplex Immunoreagents

Background

There are currently 7 known serotypes of botulinum neurotoxin (BoNT) classified upon non-cross reactivity of neutralizing immunoglobulins. Non-neutralizing immunoglobulins, however, can exhibit cross-reactivities between 2 or more serotypes, particularly mosaic forms, which can hamper the development of highly specific immunoassays, especially if based on polyclonal antisera. Here we employ facile recombinant antibody technology to subtractively select ligands to each of the 7 BoNT serotypes, resulting in populations with very high specificity for their intended serotype.

Methods and Findings

A single llama was immunized with a cocktail of 7 BoNT toxoids to generate a phage display library of single domain antibodies (sdAb, VHH or nanobodies) which were selected on live toxins. Resulting sdAb were capable of detecting both toxin and toxin complex with the best combinations able to detect 100s-10s of pg per 50 µL sample in a liquid bead array. The most sensitive sdAb were combined in a heptaplex assay to identify each of the BoNT serotypes in buffer and milk and to a lesser extent in carrot juice, orange juice and cola. Several anti-A(1) sdAb recognized A2 complex, showing that subtype cross-reactivity within a serotype was evident. Many of our sdAb could act as both captor and tracer for several toxin and toxin complexes suggesting sdAb can be used as architectural probes to indicate BoNT oligomerisation. Six of 14 anti-A clones exhibited inhibition of SNAP-25 cleavage in the neuro-2A assay indicating some sdAb had toxin neutralizing capabilities. Many sdAb were also shown to be refoldable after exposure to high temperatures in contrast to polyclonal antisera, as monitored by circular dichroism.

Conclusions

Our panel of molecularly flexible antibodies should not only serve as a good starting point for ruggedizing assays and inhibitors, but enable the intricate architectures of BoNT toxins and complexes to be probed more extensively.     

Subsets of myeloid-derived suppressor cells in tumor-bearing mice

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of cells that play a critical role in tumor associated immune suppression. In an attempt to identify a specific subset of MDSC primarily responsible for immunosuppressive features of these cells, 10 different tumor models were investigated. All models showed variable but significant increase in the population of MDSC. Variability of MDSC expansion in vivo matched closely the effect of tumor cell condition medium in vitro. MDSC consists of two major subsets of Ly6G(+)Ly6C(low) granulocytic and Ly6G(-)Ly6C(high) monocytic cells. Granulocytic MDSC have increased level of reactive oxygen species and undetectable level of NO whereas monocytic MDSC had increased level of NO but undetectable levels of reactive oxygen species. However, their suppressive activity per cell basis was comparable. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. We performed a phenotypical and functional analysis of several surface molecules previously suggested to be involved in MDSC-mediated suppression of T cells: CD115, CD124, CD80, PD-L1, and PD-L2. Although substantial proportion of MDSC expressed those molecules no differences in the level of their expression or the proportion, positive cells were found between MDSC and cells from tumor-free mice that lack immune suppressive activity. The level of MDSC-mediated T cell suppression did not depend on the expression of these molecules. These data indicate that suppressive features of MDSC is caused not by expansion of a specific subset but more likely represent a functional state of these cells.     

Dual mechanisms of regulation of Na/H exchanger NHE-3 by parathyroid hormone in rat kidney

Parathyroid hormone (PTH) is a potent inhibitor of mammalian renal proximal tubule sodium absorption via suppression of the apical membrane Na/H exchanger (NHE-3). We examined the mechanisms by which PTH inhibits NHE-3 activity by giving an acute intravenous PTH bolus to parathyroidectomized rats. Parathyroidectomy per se increased apical membrane NHE-3 activity and antigen. Acute infusion of PTH caused a time-dependent decrease in NHE-3 activity as early as 30 min. Decrease in NHE-3 activity at 30 and 60 min was accompanied by increased NHE-3 phosphorylation. In contrast to the rapid changes in NHE-3 activity and phosphorylation, decrease in apical membrane NHE-3 antigen was not detectable until 4-12 h after the PTH bolus. The decrease in apical membrane NHE-3 occurred in the absence of changes in total renal cortical NHE-3 antigen. Pretreatment of the animals with the microtubule-disrupting agent colchicine blocked the PTH-induced decrease in apical NHE-3 antigen. We propose that PTH acutely cause a decrease in NHE-3 intrinsic transport activity possibly via a phosphorylation-dependent mechanism followed by a decrease in apical membrane NHE-3 antigen via changes in protein trafficking.     

Avian fruit preferences across a Puerto Rican forested landscape: pattern consistency and implications for seed removal

Avian fruit consumption may ensure plant reproductive success when frugivores show consistent preference patterns and effectively remove and disperse seeds. In this study we examined avian fruit preferences and their seed-removal services at five study sites in north-central Puerto Rico. At each site, we documented the diet of seven common fruit-eating avian species from February to September 1998. Using foraging observations and area-based estimates of fruit abundance, we examined preference patterns of birds. We found that 7 out of 68 fleshy-fruited plant species were responsible for most of the fruit diet of birds. Seventeen plant species were preferred and four of them were repeatedly preferred across several study sites and times by at least one avian species. Preferred plant species comprised a small percentage of fleshy fruits at each site (<15% in four out of five study sites), but showed extended phenology patterns. The quantity of seeds removed by frugivore species was not strictly related to preferences. Some frugivores showing no preference could effectively remove more seeds from plants at some locations than species exhibiting constancy in their patterns of preference. Only two frugivores, Euphonia musica and Vireo altiloquous, removed most of the seeds of plants for which they exhibited repeated preference across the landscape. Preference patterns, particularly those exhibiting consistency in space and time for plant species having prolonged fruiting periods, may have important mechanistic consequences for the persistence, succession, and regeneration of tropical plant communities.     

Functional Characterization of the Purified Holo Form of Hemoglobin I from Lucina pectinata Overexpressed in Escherichia coli

The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico. It contains three different hemoglobins. Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state. Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA). Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography. Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively. The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin. Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI. The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively. The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier. This effect is attributed mostly to the first of two missense mutations [Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101] and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations. The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics. A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.     

Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate–binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.     

Sampling bees in tropical forests and agroecosystems: a review

Bees are the predominant pollinating taxa, providing a critical ecosystem service upon which many angiosperms rely for successful reproduction. Available data suggests that bee populations worldwide are declining, but scarce data in tropical regions precludes assessing their status and distribution, impact on ecological services, and response to management actions. Herein, we reviewed >150 papers that used six common sampling methods (pan traps, baits, Malaise traps, sweep nets, timed observations and aspirators) to better understand their strengths and weaknesses, and help guide method selection to meet research objectives and development of multi-species monitoring approaches. Several studies evaluated the effectiveness of sweep nets, pan traps, and malaise traps, but only one evaluated timed observations, and none evaluated aspirators. Only five studies compared two or more of the remaining four sampling methods to each other. There was little consensus regarding which method would be most reliable for sampling multiple species. However, we recommend that if the objective of the study is to estimate abundance or species richness, malaise traps, pan traps and sweep nets are the most effective sampling protocols in open tropical systems; conversely, malaise traps, nets and baits may be the most effective in forests. Declining bee populations emphasize the critical need in method standardization and reporting precision. Moreover, we recommend reporting a catchability coefficient, a measure of the interaction between the resource (bee) abundance and catching effort. Melittologists could also consider existing methods, such as occupancy models, to quantify changes in distribution and abundance after modeling heterogeneity in trapping probability, and consider the possibility of developing monitoring frameworks that draw from multiple sources of data.     

Nest occurrence and survival of King Rails in fire‐managed coastal marshes in North Carolina and Virginia

Prescribed burning maintains marsh habitat, but its impact on breeding King Rails (Rallus elegans) is poorly understood. This practice may serve as a means to enhance populations of a species whose numbers are declining in the southeastern United States. We used call‐broadcast surveys and nest searches to categorize the state of occupied plots by the presence or non‐presence of nesting activity in the Back Bay region, North Carolina and Virginia, in 2010. We also used nest video surveillance to estimate nest survival in 2009 and 2010. The probabilities that a surveyed plot was occupied (¹) and contained an active nest (²) were higher in recently burned marsh plots (0–1 year‐since‐burn [YSB]) than in plots with ≥2 YSB at Mackay Island and Back Bay National Wildlife Refuges (NWRs). Highest probabilities were recorded in 0–1 YSB plots at Mackay Island NWR ( = 0.96 ± 0.04, = 0.75 ± 0.18), and the lowest in ≥2 YSB plots at Back Bay NWR ( = 0.21 ± 0.10, = 0.03 ± 0.04). Nest survival from egg laying to hatching (31 d) was 0.48 (95% confidence interval [CI] = 0.06–0.83), with an estimated 0.79 (95% CI = 0.29–0.96) survival rate for the incubation stage (21 d). These nest survival estimates for King Rails in fire‐managed marshes were similar to estimates for other populations. Measures of vegetation cover, proxies for concealment, did not differ between nest sites and unused sites, even within recently burned marshes. This lack of differences in vegetation structure suggests that regrowth occurs rapidly during the period between burning (winter months) and the onset of reproduction (late April). Thus, recently burned marshes may benefit nesting King Rails by providing nest concealment. In addition, burned marshes may enhance availability of many invertebrates. Although we found that the probability that surveyed plots contained active nests was higher in recently burned marsh plots, estimates of fledging success are needed before marsh burns can be considered an effective means of fostering population growth.     

Thyroid receptor ligands. Part 4: 4'-amido bioisosteric ligands selective for the thyroid hormone receptor beta

Based on the examination of the X-ray crystallographic structures of the LBD of TRalpha and TRbeta in complex with KB-141 (2), a number of novel 4'-hydroxy bioisosteric thyromimetics were prepared. Optimal affinity and beta-selectivity (33 times), was found with a medium-sized alkyl-substituted amido group; iso-butyl (12c). It can be concluded that bioisosteric replacements of the 4'-hydroxy position represent a new promising class of TRbeta-selective synthetic thyromimetics.