Multiple factors interact to produce responses resembling spectrum of human disease in Campylobacter jejuni infected C57BL/6 IL-10-/- mice

Background  

Campylobacter jejuni infection produces a spectrum of clinical presentations in humans - including asymptomatic carriage, watery diarrhea, and bloody diarrhea - and has been epidemiologically associated with subsequent autoimmune neuropathies. This microorganism is genetically variable and possesses genetic mechanisms that may contribute to variability in nature. However, relationships between genetic variation in the pathogen and variation in disease manifestation in the host are not understood. We took a comparative experimental approach to explore differences among different C. jejuni strains and studied the effect of diet on disease manifestation in an interleukin-10 deficient mouse model.     

Toward Next Generation Plasma Profiling via Heat-induced Epitope Retrieval and Array-based Assays

There is a need for high throughput methods for screening patient samples in the quest for potential biomarkers for diagnostics and patient care. Here, we used a combination of undirected target selection, antibody suspension bead arrays, and heat-induced epitope retrieval to allow for protein profiling of human plasma in a novel and systematic manner. Several antibodies were found to reveal altered protein profiles upon epitope retrieval at elevated temperatures with limits of detection improving into lower ng/ml ranges. In a study based on prostate cancer patients, several proteins with differential profiles were discovered and subsequently validated in an independent cohort. For one of the potential biomarkers, the human carnosine dipeptidase 1 protein (CNDP1), the differences were determined to be related to the glycosylation status of the targeted protein. The study shows a path of pursuit for large scale screening of biobank repositories in a flexible and proteome-wide fashion by utilizing heat-induced epitope retrieval and using an antibody suspension bead array format.There is a great need for protein biomarkers for early diagnosis of disease as well as for prognostic markers in which the outcome of a particular disease or treatment can be predicted (1). In particular, biomarkers that make it possible to monitor the progress of treatment or the reoccurrence of a particular disease are of great clinical value. However, there are still few protein biomarkers in clinical practice today, and despite many biomarker discovery efforts by many laboratories using many different approaches, a limited number have been introduced into the clinical routine during the last 10 years (2). The complexity of serum or plasma proteomes with their broad dynamic range of protein concentrations and the lack of high throughput methods with high sensitivity have hampered such discovery and validation efforts.The most common approach for protein biomarker discovery today is the use of proteomics methods in which samples from case-control groups are compared using biochemical and biophysical methods, most notably with mass spectrometry (3). The introduction of more and more sophisticated instrumentation has increased the sensitivity and throughput of mass spectrometry during the last years (4). One of the advantages with mass spectrometry is that the method also allows for the detection of differences in protein modifications, such as glycosylation or phosphorylation, which have been found useful for some applications (5). Although many potential biomarkers have been discovered using mass spectrometry, the approach is yet limited to the analysis of a relatively small number of patient samples.The alternative approach for biomarker discovery is to use affinity probes, usually antibodies but also other reagents, such as aptamers (6) or Affibody molecules (7). The advantage of such probe-based methods is the possibility to analyze many samples in parallel, and many assays based on antibodies, such as ELISA, are very sensitive in the sub-ng/ml range. In particular, sandwich immunoassays in which two separate antibodies are used to increase the sensitivity and selectivity allow proteins to be assayed down to pg/ml (8). Recently, new assays based on amplification methods have been described, such as the proximity ligation method (9), and these have the potential to score protein on a single molecule level. However, the lack of validated antibodies to most human proteins (10) makes it impossible to use antibody-based protocols for a majority of the potential protein targets, and this is even more difficult for assays based on paired antibodies that require two distinct antibodies with separate and non-overlapping epitopes. Because of this limitation, current studies are directed by candidate target lists reported in the literature (11) or in associated gene expression studies (12) or built on collections of in-house binder libraries (13).Recently, new efforts have been described for the generation of antibodies on a whole-proteome level (14). Version 6 of the Human Protein Atlas contains validated antibodies toward proteins from 8,400 human genes, corresponding to 42% of the protein-encoded genes in man. All antibodies published in the Human Protein Atlas are publicly available and include a total of more than 40 antibody providers from the United States, Canada, Europe, Australia, and Asia. Several other efforts, such as the ProteomeBinder (15), the SH2 consortium (16), and the NCI affinity capture project (17), have recently been initiated with the aim to generate affinity reagents toward human protein targets. The objective of these efforts is to have publicly available antibodies to a representative protein from all of the protein-encoded genes by 2014 (18), and this emphasizes the need to develop high throughput methods for immunobased protein profiling to leverage this tool box of antibodies to allow high throughput biomarker discovery.We have shown earlier that antibodies utilized in suspension bead arrays can be used for profiling proteins in serum and plasma (19). Hereby, we found that the ability to detect proteins such as components of the complement system was enhanced by heat treatment, most likely because epitopes might be exposed at elevated temperatures and thus become available for antibody binding. In particular, this is likely to be the case for antibodies recognizing linear epitopes on the protein target. Here, we analyzed the functionality of antibodies following different epitope retrieval protocols at different temperatures, and we describe a method for multiplex analysis of plasma or serum using plasma from patients with elevated PSA1 levels as an example. A method suitable for analysis of large numbers of biobank samples is presented.     

Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.     

The 6th HUPO Antibody Initiative (HAI) Workshop: Sharing Data About Affinity Reagents and Other Recent Developments September 2009, Toronto,Canada

The Human Antibody Initiative (HAI) aims to promote and facilitate the use of antibodies for proteomics research. The 6th workshop for the HUPO Antibody Initiative (HAI) held in September 2009 was co‐chaired by Michael Snyder and Mathias Uhlen and discussed several aspects of antibody production, their validation, and attempts to standardise this process, in particular, when subsequently described in the literature. An update on the progress of the Human Protein Atlas was also presented to the attendees.     

Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods

Centrosomes in animal cells are dynamic organelles with a proteinaceous matrix of pericentriolar material assembled around a pair of centrioles. They organize the microtubule cytoskeleton and the mitotic spindle apparatus. Mature centrioles are essential for biogenesis of primary cilia that mediate key signalling events. Despite recent advances, the molecular basis for the plethora of processes coordinated by centrosomes is not fully understood. We have combined protein identification and localization, using PCP-SILAC mass spectrometry, BAC transgeneOmics, and antibodies to define the constituents of human centrosomes. From a background of non-specific proteins, we distinguished 126 known and 40 candidate centrosomal proteins, of which 22 were confirmed as novel components. An antibody screen covering 4000 genes revealed an additional 113 candidates. We illustrate the power of our methods by identifying a novel set of five proteins preferentially associated with mother or daughter centrioles, comprising genes implicated in cell polarity. Pulsed labelling demonstrates a remarkable variation in the stability of centrosomal protein complexes. These spatiotemporal proteomics data provide leads to the further functional characterization of centrosomal proteins.     

The influence of water level on macrophyte growth and trophic interactions in eutrophic Mediterranean shallow lakes: a mesocosm experiment with and without fish

1. Water‐level fluctuations are typical of lakes located in the semi‐arid Mediterranean region, which is characterised by warm rainy winters and hot dry summers. Ongoing climate change may exacerbate fluctuations and lead to more severe episodes of drought, so information on the effects of water level on the functioning of lake ecosystems in such regions is crucial. 2. In eutrophic Lake Eymir, Turkey, we conducted a 4‐month (summer) field experiment using cylindrical 0.8‐m‐ (low‐water‐level) and 1.6‐m‐deep (high‐water‐level) mesocosms (kept open to the sediment and atmosphere). Fish (tench, Tinca tinca, and bleak, Alburnus escherichii) were added to half of the mesocosms, while the rest were kept fishless. Ten shoots of Potamogeton pectinatus were transplanted to each mesocosm. 3. Sampling for physicochemical variables, chlorophyll a (chl‐a), zooplankton and per cent plant volume inhabited (PVI%) by macrophytes was conducted weekly during the first 5 weeks, and subsequently biweekly. Macrophytes were harvested on the last sampling date. During the course of the experiment, the water level decreased by 0.41 ± 0.06 m. 4. Throughout the experiment, fish affected zooplankton abundance (?), nutrient concentrations (+), chl‐a (+) and water clarity (?) most strongly in the low‐water‐level mesocosms and the zooplankton community shifted towards dominance of small‐sized forms. The fishless mesocosms had a higher zooplankton/phytoplankton ratio, suggesting higher grazing. 5. Greatest macrophyte growth was observed in the low‐water‐level fishless mesocosms. However, despite high nutrient concentrations and low water clarity, macrophytes were also abundant in the fish mesocosms and particularly increased following a water‐level decrease from midsummer onwards. Macrophyte growth was poor in the high‐water‐level mesocosms, even in the fishless ones with high water clarity. This was ascribed to extensive periphyton development reducing light availability for the macrophytes. 6. Our results indicate that a reduction in water level during summer may help maintain the growth of macrophytes in Mediterranean eutrophic shallow lakes, despite a strong negative effect of fish predation on water clarity. It is therefore probable that an expected negative effect of global climate change on water clarity because of eutrophication and enhanced top‐down control of fish may be, at least partly, counteracted by reduced water level, provided that physical disturbance is not severe.     

Nifedipine loaded chitosan microspheres prepared by emulsification phase-separation

A high yield of nifedipine-chitosan microspheres could be obtained using an emulsification phase-separation method. A high level of entrapment of nifedipine in the microspheres was achieved. The microspheres exhibited excellent swelling properties. Differential scanning calorimetry, X-ray diffractometry, and scanning electron microscopy confirmed that at 1.84% loading, nifedipine was dispersed molecularly. The microspheres exhibited faster release at low loadings compared to high loadings. Fitting the data to the coupled Fickian/case II equation, showed that at low loadings polymer relaxation coefficients (k₂) were high. As the polymer content increased in the microspheres, the value of n (diffusional exponent characteristic of the release mechanism) approached one, which is indicative of zero order.     

Real time in vitro studies of doxorubicin release from PHEMA nanoparticles

Background  

Many anticancer agents have poor water solubility and therefore the development of novel delivery systems for such molecules has received significant attention. Nanocarriers show great potential in delivering therapeutic agents into the targeted organs or cells and have recently emerged as a promising approach to cancer treatments. The aim of this study was to prepare and use poly-2-hydroxyethyl methacrylate (PHEMA) nanoparticles for the controlled release of the anticancer drug doxorubicin.