Antibody suspension bead arrays within serum proteomics

Antibody microarrays offer a powerful tool to screen for target proteins in complex samples. Here, we describe an approach for systematic analysis of serum, based on antibodies and using color-coded beads for the creation of antibody arrays in suspension. This method, adapted from planar antibody arrays, offers a fast, flexible, and multiplexed procedure to screen larger numbers of serum samples, and no purification steps are required to remove excess labeling substance. The assay system detected proteins down to lower picomolar levels with dynamic ranges over 3 orders of magnitude. The feasibility of this workflow was shown in a study with more than 200 clinical serum samples tested for 20 serum proteins.     

The epitope space of the human proteome

In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteome-wide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed.     

Using Small-Scale Studies to Prioritize Threats and Guide Recovery of a Rare Hemiparasitic Plant: Cordylanthus rigidus ssp. littoralis

Background

Recovering endangered species would benefit from identifying and ranking of the factors that threaten them. Simply managing for multiple positive influences will often aid in recovery; however, the relative impacts of multiple threats and/or interactions among them are not always predictable. We used a series of experiments and quantitative observational studies to examine the importance of five potential limiting factors to the abundance of a state-listed endangered hemiparasitic annual forb, Cordylanthus rigidus ssp. littoralis (C.r.l., California, USA): host availability, mammalian herbivores, insect seed predators, fire suppression, and exotic species. While this initial assessment is certainly not a complete list, these factors stem from direct observation and can inform provisional recommendations for management and further research.

Methodology and Principal Findings

Studies were conducted at five sites and included assessments of the influence of host availability, exotic species, exclusion of mammalian herbivores and insect seed predators on C.r.l. productivity, and simulated effects of fire on seed germination. C.r.l. was limited by multiple threats: individuals with access to host species produced up to three times more inflorescences than those lacking hosts, mammalian herbivory reduced C.r.l. size and fecundity by more than 50% and moth larvae reduced seed production by up to 40%. Litter deposition and competition from exotic plant species also appears to work in conjunction with other factors to limit C.r.l. throughout its life cycle.

Conclusions and Significance

The work reported here highlights the contribution that a series of small-scale studies can make to conservation and restoration. Taken as a whole, the results can be used immediately to inform current management and species recovery strategies. Recovery of C.r.l. will require management that addresses competition with exotic plant species, herbivore pressure, and availability of preferred host species.     

Two distinctly different sperm storage organs in female Dysdera erythrina (Araneae: Dysderidae)

The haplogyne spider D. erythrina possesses two distinctly different sperm storage organs: a bilobed anterior spermatheca and a large, sac-like posterior diverticulum. The glandular equipment of both storage types is markedly different: the glandular tissue of the spermatheca is composed of complicated glandular units comprising a cuticular ductule and three canal cells (class 3 cells) whereas the glandular tissue of the posterior diverticulum is composed of simple gland cells that discharge their product through the cuticle (class 1 cells). Thus, the glandular products produced differ, leading to different storage conditions for the spermatozoa from copulation to egg laying. It is suggested that multiple organ types have evolved to facilitate specialization in short-term and long-term storage and to allow (posterior diverticulum) or prevent (spermatheca) males from accessing previously stored sperm.     

Calcium signaling in placenta

The placenta sustains the developing fetus throughout gestation and its major functions include nutrition, gas and waste exchange via a variety of passive or active mechanisms. Up to 30 g of calcium (Ca(2+)) actively crosses the trophoblast layer during human pregnancy. The Ca(2+) ion not only plays an important role for skeletal development but is also an essential second messenger. This review is intended to highlight the implications of Ca(2+) signaling during reproduction and specifically placentation. Initially, a Ca(2+) wave induces fertilization of the oocyte. The intracellular Ca(2+) concentration is key for the blastocyst implantation, proper placental development and function. Current knowledge of many proteins involved in placental Ca(2+) regulation and their function in pathologic conditions is largely limited. Recent studies, however, point to alterations in Ca(2+) homeostasis in placental pathologies such as pre-eclampsia (PE) and intrauterine growth restriction (IUGR). A broader understanding of the role of Ca(2+) signaling during human reproduction may offer insight into impaired pregnancy outcomes.     

A human protein atlas for normal and cancer tissues based on antibody proteomics

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, approximately 400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.     

Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy

We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.     

The role of TGF-beta1 as a determinant of foreign body reaction to alloplastic materials in rat fibroblast cultures: comparison of different commercially available polypropylene meshes for hernia repair

BACKGROUND: Animal experiments on hernia repair demonstrated better biocompatibility of light-weight polypropylene meshes. However, implanted medical devices trigger a variety of adverse tissue responses, such as inflammation, fibrosis, infection and thrombosis, but the mechanisms involved in such responses remain largely unknown. This study aimed to determine the effect of transforming growth factor beta1 (TGF-beta1) on host tolerance by quantification of foreign body reaction in cultured fibroblasts depending on the amount and composition of implanted material used for hernia repair. MATERIALS AND METHODS: An NRK-49F fibroblast culture was incubated in the presence of 4 commercially available meshes approved for hernia repair. A mesh-free cell suspension served as a control group, in which the influence of TGF-beta1 on fibroblasts was evaluated. Levels of TGF-beta1 in the supernatant were dynamically measured in a time interval of 6 to 96 h and cell proliferation rates were assessed colorimetrically using MTT test. RESULTS: A dose-dependent suppression of fibroblasts proliferation by TGF-beta1 was observed. All meshes suppressed the secretion of TGF-beta1 and conversely increased significantly cell proliferation in comparison to the control group (p<0.01) in the first 24 to 48 h of incubation. That effect was more pronounced in meshes partially containing absorbable material when compared to samples of pure polypropylene meshes (p<0.05) and to the control group (p<0.001). CONCLUSION: Our experiment revealed that early biological reaction of connective tissue cells towards polypropylene meshes and their variants depended much more on the composition and type of the material than on its absolute amount. The assumption that material weight reduction alone might affect the foreign body reaction of mesh implants could not be confirmed by our in vitro study.