Affinity ligand selection from a library of small molecules: assay development, screening, and application

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.     

Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.     

Familial aggregation in lone atrial fibrillation

Atrial fibrillation (AF) is the most common clinical arrhythmia and a major risk factor for stroke. To investigate the role of genetic factors in a typical clinical population, we determined the extent of familial aggregation in patients with lone AF. To estimate the relative risk to family members, the prevalence of AF for each class of relative was compared to the prevalence in the comparable age and sex group from the general population. Family members had an increased relative risk of AF compared to the general population (risk ratio; 95% confidence intervals): sons (8.1; 2.0–32), daughters (9.5; 1.3–67), brothers (70; 47–102), sisters (34; 14–80), mothers (4.0; 2.5–6.5) and fathers (2.0; 1.2–3.6). Relatives of probands with lone AF are at a substantially increased risk of developing this arrhythmia suggesting a Mendelian genetic contribution to the etiology of this common trait.     

The effect of grazing and nutrient supply on periphyton associated bacteria

The effects of nutrient additions and grazing by macro-invertebrates on periphyton-associated algae and bacteria were studied by performing an enclosure experiment on three occasions from early spring to summer at mesotrophic Lake Erken and V?dd?, at the Swedish Baltic coast. There were significant interactions between nutrient additions and grazing on bacterial biomass and specific activity in Lake Erken. Thus, the importance of either bottom-up or top-down effects could not be singled out. Bacterial biomass increased with enrichment only in the absence of grazers. Grazer presence tended to increase bacterial biomass in ambient nutrient conditions, but to decrease bacterial biomass under enrichment. For specific activity the positive response to enrichment was restricted to grazer presence. Hence, grazing by macro-invertebrates may have an indirect positive effect on bacterial activity by enhancing nutrient conditions through their feeding activities and/or fecal pellets production. In addition, we found a significant relationship between bacterial production and chlorophyll a at both sites. This relationship weakened in the presence of macro-invertebrates. Thus, the importance of internal nutrient regeneration by bacteria and algae decreased, possibly due to increased nutrient availability, in the presence of macro-invertebrate grazers.     

Insights into the evolution of freshwater sponges (Porifera: Demospongiae: Spongillina): Barcoding and phylogenetic data from Lake Tanganyika endemics indicate multiple invasions and unsettle existing taxonomy

Sponges are a conspicuous element in many benthic habitats including in Africa's oldest, deepest lake, Lake Tanganyika. Despite their prevalence and pivotal ecological role as filter feeders, knowledge of the evolutionary history of sponges is in its infancy. Here, we provide the first molecular analysis targeting the evolution of sponges from Lake Tanganyika. Independent markers indicate the occurrence of several colonisation events which have shaped the current Tanganyikan lacustrine sponge biodiversity. This is in contrast to a range of previously studied organisms that have diversified within the lake from single lineages. Our tree reconstructions indicate the presence of two genera, Oncosclera and Eunapius, which are globally distributed. Therefore, we reject the hypothesis of monophyly for the sponges from Lake Tanganyika and challenge existing higher taxonomic structure for freshwater sponges.     

Concomitant Tumor Expression of EGFR and TATI/SPINK1 Associates with Better Prognosis in Colorectal Cancer

Background

Epidermal growth factor receptor (EGFR) activation plays a role in colorectal cancer (CRC) carcinogenesis, and anti-EGFR drugs are used in treatment of advanced CRC. One of the EGFR ligands is tumor-associated trypsinogen inhibitor TATI, also called serine protease inhibitor Kazal type1 (SPINK 1), which we recently showed to be an independent prognostic marker in CRC.

Methods

We studied the prognostic value of immunohistochemical expression of EGFR and concomitant expression of EGFR and TATI/SPINK1 in a series of 619 colorectal cancer patients.

Results

Of the samples, 92% were positive for EGFR. EGFR+/TATI+ was seen in 62.8%, EGFR+/TATI− in 29.5%, EGFR−/TATI+ in 4.9%, and EGFR−/TATI− in 2.7% of patients. EGFR expression correlated with WHO grade (p = 0.040). In univariate analysis, EGFR expression correlated with favourable survival (p = 0.006). EGFR+/TATI+ patients showed better survival than did those with other combinations (p<0.001). In multivariate analysis, EGFR+/TATI+ was an independent prognostic factor of favourable prognosis (p<0.001).

Conclusion

Concomitant positivity of EGFR and TATI/SPINK1 predicts favourable prognosis in CRC.     

Fibroblast growth factors and their receptors in cancer

FGFs (fibroblast growth factors) and their receptors (FGFRs) play essential roles in tightly regulating cell proliferation, survival, migration and differentiation during development and adult life. Deregulation of FGFR signalling, on the other hand, has been associated with many developmental syndromes, and with human cancer. In cancer, FGFRs have been found to become overactivated by several mechanisms, including gene amplification, chromosomal translocation and mutations. FGFR alterations are detected in a variety of human cancers, such as breast, bladder, prostate, endometrial and lung cancers, as well as haematological malignancies. Accumulating evidence indicates that FGFs and FGFRs may act in an oncogenic fashion to promote multiple steps of cancer progression by inducing mitogenic and survival signals, as well as promoting epithelial-mesenchymal transition, invasion and tumour angiogenesis. Therapeutic strategies targeting FGFs and FGFRs in human cancer are therefore currently being explored. In the present review we will give an overview of FGF signalling, the main FGFR alterations found in human cancer to date, how they may contribute to specific cancer types and strategies for therapeutic intervention.     

Amassing diversity in an ancient lake: evolution of a morphologically diverse parthenogenetic gastropod assemblage in Lake Malawi

Exceptional ecological niche diversity, clear waters and unique divergent selection pressures have often been invoked to explain high morphological and genetic diversity of taxa within ancient lakes. However, it is possible that in some ancient lake taxa high diversity has arisen because these historically stable environments have allowed accumulation of lineages over evolutionary timescales, a process impossible in neighbouring aquatic habitats undergoing desiccation and reflooding. Here we examined the evolution of a unique morphologically diverse assemblage of thiarid gastropods belonging to the Melanoides polymorpha'complex' in Lake Malawi. Using mitochondrial DNA sequences, we found this Lake Malawi complex was not monophyletic, instead sharing common ancestry with Melanoides anomala and Melanoides mweruensis from the Congo Basin. Fossil calibrations of molecular divergence placed the origins of this complex to within the last 4 million years. Nuclear amplified fragment length polymorphism markers revealed sympatric M. polymorpha morphs to be strongly genetically differentiated lineages, and males were absent from our samples indicating that reproduction is predominantly parthenogenetic. These results imply the presence of Lake Malawi as a standing water body over the last million years or more has facilitated accumulation of clonal morphological diversity, a process that has not taken place in more transient freshwater habitats. As such, the historical stability of aquatic environments may have been critical in determining present spatial distributions of biodiversity.     

Influence of bioreactor scale and complex medium on probing control of glucose feeding in cultivations of recombinant strains of Escherichia coli

The objective of this work was to evaluate the performance of a feedback glucose control strategy (the probing strategy) in production relevant bioreactors with complex and mineral media. Experimental results from fed-batch cultivations with two recombinant Escherichia coli constructs expressing two different human therapeutic proteins were used to assess the performance and limitations of the glucose probing technique. Even though the performance of the probing strategy was affected by scale and complex media, this methodology rapidly identified a glucose feed protocol similar to an experimentally derived feed regime. This methodology may serve as a powerful tool for industrial process development and in optimization of glucose feed regimes when transferring process technology from one bioreactor system to another.