Ionome and elemental transport kinetics shaped by parallel evolution in threespine stickleback

Evidence that organisms evolve rapidly enough to alter ecological dynamics necessitates investigation of the reciprocal links between ecology and evolution. Data that link genotype to phenotype to ecology are needed to understand both the process and ecological consequences of rapid evolution. Here, we quantified the suite of elements in individuals (i.e., ionome) and differences in the fluxes of key nutrients across populations of threespine stickleback. We find that allelic variation associated with freshwater adaptation that controls bony plating is associated with changes in the ionome and nutrient recycling. More broadly, we find that adaptation of marine stickleback to freshwater conditions shifts the ionomes of natural populations and populations raised in common gardens. In both cases ionomic divergence between populations was primarily driven by differences in trace elements rather than elements typically associated with bone. These findings demonstrate the utility of ecological stoichiometry and the importance of ionome‐wide data in understanding eco‐evolutionary dynamics.     

Comprehensive manipulation of glycosylation profiles across development scales

The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.     

Inhibition of the dopamine D1 receptor signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.     

The role of the fibronectin IGD motif in stimulating fibroblast migration

The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 10(6) times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN.     

Identification of novel high-frequency DNA methylation changes in breast cancer

Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200), a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.