Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF·c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSF_M) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSF_M variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSF_M structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSF_M and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSF_M residues critical for M-CSF·c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF·c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.     

Soluble minerals in chemical evolution

The adsorption of 5-AMP onto solid CaSO₄ · 2H₂O was studied in a saturated suspension as a function of pH and electrolyte concentration. The adsorption is pH-dependent and is directly correlated with the charge on the 5-AMP molecule which is determined by the state of protonation of the N-1 nitrogen of the purine ring and the phosphate oxygens. It is proposed that the binding that occurs between the nucleotide and the salt is electrostatic in nature. The adsorption decreases with increasing ionic strength of the solution which means that in a fluctuating environment of wetting and drying cycles, a biomolecule similar to 5-AMP could be expected to desorb during the drying phase. The results indicate that CaSO₄ · 2H₂O can serve as a concentrating surface for biomolecules. The significance of this is discussed with regard to the possible role of soluble minerals and their surfaces in a geochemical model consistent with the evolution of the Earth and the origin of life.     

Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.     

The effects of proteasomal inhibition on synaptic proteostasis

Synaptic function crucially depends on uninterrupted synthesis and degradation of synaptic proteins. While much has been learned on synaptic protein synthesis, little is known on the routes by which synaptic proteins are degraded. Here we systematically studied how inhibition of the ubiquitin‐proteasome system (UPS) affects the degradation rates of thousands of neuronal and synaptic proteins. We identified a group of proteins, including several proteins related to glutamate receptor trafficking, whose degradation rates were significantly slowed by UPS inhibition. Unexpectedly, however, degradation rates of most synaptic proteins were not significantly affected. Interestingly, many of the differential effects of UPS inhibition were readily explained by a quantitative framework that considered known metabolic turnover rates for the same proteins. In contrast to the limited effects on protein degradation, UPS inhibition profoundly and preferentially suppressed the synthesis of a large number of synaptic proteins. Our findings point to the importance of the UPS in the degradation of certain synaptic proteins, yet indicate that under basal conditions most synaptic proteins might be degraded through alternative pathways.     

Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next‐generation sequencing to allow such large‐scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large‐scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost‐effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.     

Modeling the effects of enclosure size on geometry learning

Several recent studies have shown that chickens, fish, and humans trained to find a reward in a corner of a rectangular enclosure with distinctive features rely more on the geometry of the enclosure in small enclosures and rely more on the features in large enclosures. Here, these results are modeled using a recent associative model of geometry learning [Miller, N.Y., Shettleworth, S.J., 2007. Learning about environmental geometry: an associative model. J. Exp. Psychol. Anim. B 33, 191–212]. By adjusting the salience of either geometric or featural information or both the model is capable of reproducing much of the data on the effects of enclosure size on geometry learning.     

Group Selection and Contribution of Minority Variants during Virus Adaptation Determines Virus Fitness and Phenotype

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.