Gene expression actualizes the organismal phenotypes encoded within the genome in an environment-dependent manner. Among all encoded phenotypes, cell population growth rate (fitness) is perhaps the most important, since it determines how well-adapted a genotype is in various environments. Traditional biological measurement techniques have revealed the connection between the environment and fitness based on the gene expression mean. Yet, recently it became clear that cells with identical genomes exposed to the same environment can differ dramatically from the population average in their gene expression and division rate (individual fitness). For cell populations with bimodal gene expression, this difference is particularly pronounced, and may involve stochastic transitions between two cellular states that form distinct sub-populations. Currently it remains unclear how a cell population's growth rate and its subpopulation fractions emerge from the molecular-level kinetics of gene networks and the division rates of single cells. To address this question we developed and quantitatively characterized an inducible, bistable synthetic gene circuit controlling the expression of a bifunctional antibiotic resistance gene in Saccharomyces cerevisiae. Following fitness and fluorescence measurements in two distinct environments (inducer alone and antibiotic alone), we applied a computational approach to predict cell population fitness and subpopulation fractions in the combination of these environments based on stochastic cellular movement in gene expression space and fitness space. We found that knowing the fitness and nongenetic (cellular) memory associated with specific gene expression states were necessary for predicting the overall fitness of cell populations in combined environments. We validated these predictions experimentally and identified environmental conditions that defined a “sweet spot” of drug resistance. These findings may provide a roadmap for connecting the molecular-level kinetics of gene networks to cell population fitness in well-defined environments, and may have important implications for phenotypic variability of drug resistance in natural settings.

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Fertilization releases the meiotic arrest and initiates the events that prepare the egg for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phosphoregulation is still unknown. We examined absolute protein and phosphosite dynamics of the fertilization response by mass spectrometry-based proteomics in electroactivated eggs. To do this, we developed an approach for calculating the stoichiometry of phosphosites from multiplexed proteomics that is compatible with dynamic, stable, and multisite phosphorylation. Overall, the data suggest that degradation is limited to a few low-abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium-activated kinases. We identify putative degradation targets and components of the slow block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

SummaryFertilization triggers release from meiotic arrest and initiates events that prepare for the ensuing developmental program. Protein degradation and phosphorylation are known to regulate protein activity during this process. However, the full extent of protein loss and phospho-regulation is still unknown. We examined absolute protein and phospho-site dynamics after fertilization by mass spectrometry-based proteomics. To do this, we developed a new approach for calculating the stoichiometry of phospho-sites from multiplexed proteomics that is compatible with dynamic, stable and multi-site phosphorylation. Overall, the data suggest that degradation is limited to a few low abundance proteins. However, this degradation promotes extensive dephosphorylation that occurs over a wide range of abundances during meiotic exit. We also show that eggs release a large amount of protein into the medium just after fertilization, most likely related to the blocks to polyspermy. Concomitantly, there is a substantial increase in phosphorylation likely tied to calcium activated kinases. We identify putative degradation targets as well as new components of the block to polyspermy. The analytical approaches demonstrated here are broadly applicable to studies of dynamic biological systems.

Evolution by natural selection is the driving force behind the endless variation we see in nature, yet our understanding of how changes at the molecular level give rise to different phenotypes and altered fitness at the population level remains inadequate. The reproductive fitness of an organism is the most basic metric that describes the chance that an organism will succeed or fail in its environment and it depends upon a complex network of inter-and intramolecular interactions. A deeper understanding of the quantitative relationships relating molecular evolution to adaptation, and consequently fitness, can guide our understanding of important issues in biomedicine such as drug resistance and the engineering of new organisms with applications to biotechnology. We have developed the "weak link" approach to determine how changes in molecular structure and function can relate to fitness and evolutionary outcomes. By replacing adenylate kinase ͑AK͒, an essential gene, in a thermophile with a homologous AK from a mesophile we have created a maladapted weak link that produces a temperature-sensitive phenotype. The recombinant strain adapts to nonpermissive temperatures through point mutations to the weak link that increase both stability and activity of the enzyme AK at higher temperatures. Here, we propose a fitness function relating enzyme activity to growth rate and use it to create a dynamic model of a population of bacterial cells. Using metabolic control analysis we show that the growth rate exhibits thresholdlike behavior, saturating at high enzyme activity as other reactions in the energy metabolism pathway become rate limiting. The dynamic model accurately recapitulates observed evolutionary outcomes. These findings suggest that in vitro enzyme kinetic data, in combination with metabolic network analysis, can be used to create fitness functions and dynamic models of evolution within simple metabolic systems. © 2010 American Institute of Physics. ͓doi:10.1063/1.3453623͔ Adaptation is the essence of Darwin's brilliant conception of natural selection and is the mechanism by which all organisms are shaped by their environment. Adaptation is a fundamental property of all life and is responsible for recent increases in the populations of drug resistant pathogens that directly impact human health. While microorganisms have relatively simpler and smaller genomes than humans, they still contain thousands of genes that interact with each other to create large networks whose response to challenges is remarkably complex and largely unpredictable. The genome defines the ways in which an organism can respond to its environment and is also a record of adaptation that encodes the "current state" of the organism. The genome provides an excellent system for recording the steps of adaptation at a molecular level (molecular evolution). The challenge of understanding molecular evolution can be addressed through the development of robust experimental systems that afford the opportunity to build and validate models for adaptation....

Wnt11 family proteins are ligands that activate a type of Dishevelled-mediated, non-canonical Wnt signaling pathway. Loss of function causes defects in gastrulation and/or anterior-posterior axis extension in all vertebrates. Non-mammalian vertebrate genomes encode two Wnt11 family proteins whose distinct functions have been unclear. We knocked down zygotic Wnt11b and Wnt11, separately and together, in Xenopus laevis. Single morphants exhibited very similar phenotypes of delayed blastopore closure, but they had different phenotypes at the tailbud stage. In response to their very similar gastrulation phenotypes, we chose to characterize dual morphants. Using dark field illuminated time-lapse imaging and kymograph analysis, we identified a failure of dorsal blastopore lip maturation that correlated with slower blastopore closure and failure to internalize the endoderm at the dorsal blastopore lip. We connected these externally visible phenotypes to cellular events in the internal tissues – including the archenteron – by imaging intact embryos stained for anillin and microtubules. The cleavage furrow protein anillin provided an exceptional cytological marker for blastopore lip and archenteron morphogenesis and the consequent disruption through loss of Wnt11 signaling. These cytological changes suggest a novel role for the regulation of contractility and stiffness of the epithelial cells that result in dramatic shape changes and are important in gastrulation.

Vertebrate development from an egg to a complex multi-tissue million-cell organism is supported by multiple phases of genome-scale remodeling of the repertoire of proteins and their post-translational modifications, yet so far we know little about these phases. In this paper we present comprehensive characterization of these processes reflected by eleven time points, approximately eleven thousand proteins, and six thousand phospho-forms in two replicates. We find that the most dramatic changes to the proteome occur during the transition to functional organ systems, which occurs as the embryo becomes a tadpole. At that time the absolute amount of non-yolk protein increases two-fold, and there is a shift in the balance of expression from proteins regulating gene expression to receptors, ligands, and proteins involved in cell-cell and cell-environment interactions. Between the early and late tadpole, the median increase of membrane and secreted proteins is substantially higher than that of nuclear proteins. For the first time, we have included quantitative phospho-proteomic data across the same developmental stages. In contrast to the significant protein changes that are concentrated at the end of the time series, the most significant phosphorylation changes are concentrated in the very early stages of development. A clear exception are phosphorylations of proteins involved in gene expression; these increase just after fertilization, with patterns that are highly correlated with the underlying protein changes. To improve our interpretation of this unique data set, we created a pipeline for identifying homologous human phosphorylations from the measured Xenopus phospho-proteome, and we estimated, where possible, the occupancy of phosphorylation sites. Overall, we detected many profound temporal transitions, which suggest concerted changes in developmental strategies in the embryo that are particularly pronounced once early patterning and specification are complete.

Despite recent progress, Malawi continues to perform poorly on key health indicators such as child mortality and life expectancy. These problems are exacerbated by a severe lack of access to health care. Health Surveillance Assistants (HSAs) help bridge this gap by providing community-level access to basic health care services. However, the success of these HSAs is limited by a lack of supplies and long distances between HSAs and patients. To address this issue, we used large-scale weighted p-median and capacitated facility location problems to create a scalable, three-tiered plan for optimal allocation of HSAs, HSA designated medical backpacks, and backpack resupply centers. Our analysis uses real data on the location and characteristics of hospitals, health centers, and the general population. In addition to offering specific recommendations for HSA, backpack, and resupply center locations, it provides general insights into the scope of the proposed HSA backpack program scale-up. In particular, it demonstrates the importance of local health centers to the resupply network. The proposed assignments are robust to changes in the underlying population structure, and could significantly improve access to medical supplies for both HSAs and patients.