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Current influenza vaccines do not elicit broadly protective immune responses against multiple strains. New strategies to focus the humoral immune response to conserved regions on influenza antigens are therefore required for recognition by broadly neutralizing antibodies. It has been suggested that B-cells with receptors that recognize conserved epitopes would be preferentially stimulated through avidity effects by mosaic particles presenting multiple forms of a variable antigen. We adapted SpyCatcher-based platforms, AP205 virus-like particles (VLPs) and mi3 nanoparticles (NPs), to covalently co-display SpyTagged hemagglutinin (HA) trimers from group 1 and group 2 influenza A strains. Here we show successful homotypic and heterotypic conjugation of up to 8 different HA trimers to both VLPs and NPs. We characterized the HA-VLPs and HA-NPs by cryo-electron tomography to derive the average number of conjugated HAs and their separation distances on particles, and compared immunizations of mosaic and homotypic particles in wild-type mice. Both types of HA particles elicited strong antibody responses, but the mosaic particles did not consistently elicit broader immune responses than mixtures of homotypic particles. We conclude that covalent attachment of HAs from currently-circulating influenza strains represents a viable alternative to current annual influenza vaccine strategies, but in the absence of further modifications, is unlikely to represent a method for making a universal influenza vaccine., Competing Interests: The authors have declared that no competing interests exist.

As demands on agriculture increase, food producers will need to employ management strategies that not only increase yields but reduce environmental impacts. Modeling is a powerful tool for informing decision-making about current and future practices. We present a model to evaluate the effects of crop diversification on the robustness of simulated farms under labor shocks. We use an example inspired by the Florida production system of high-value, labor-intensive fruits. We find that crop diversification to high-value crops is a robust strategy when labor shocks are mild, and that crop diversification becomes less valuable as more simulated farms practice it. Based on our results, we suggest that crop diversification is a useful management strategy under specific conditions, but that policies designed to encourage crop diversification must consider broad effects as well as farm-level benefits., Competing Interests: The authors have declared that no competing interests exist.

Evolutionary studies often assume that energy is the primary resource (i.e. "currency") at the heart of the survival-reproduction trade-off, despite recent evidence to the contrary. The evolutionary consequences of having a single trade-off currency versus multiple competing currencies are unknown. Using simulations, we modeled the evolution of either a single physiological currency between reproduction and survival, or of multiple such currencies. For a wide array of model specifications varying functional forms and strengths of the trade-offs, we show that the presence of multiple currencies (e.g. nutrients, time) generally results in the evolution of higher lifetime reproductive success through partial circumvention of such trade-offs. Evolution of the underlying physiology is also more highly contingent with multiple currencies. These results challenge the paradigm of a single survival-reproduction trade-off as central to life history evolution, suggesting greater roles for physiological constraints and contingency, and implying potential selection for evolution of multiple trade-off currencies.

Physiological dysregulation may underlie aging and many chronic diseases, but is challenging to quantify because of the complexity of the underlying systems. Recently, we described a measure of physiological dysregulation, DM, that uses statistical distance to assess the degree to which an individual's biomarker profile is normal versus aberrant. However, the sensitivity of DM to details of the calculation method has not yet been systematically assessed. In particular, the number and choice of biomarkers and the definition of the reference population (RP, the population used to define a "normal" profile) may be important. Here, we address this question by validating the method on 44 common clinical biomarkers from three longitudinal cohort studies and one cross-sectional survey. DMs calculated on different biomarker subsets show that while the signal of physiological dysregulation increases with the number of biomarkers included, the value of additional markers diminishes as more are added and inclusion of 10-15 is generally sufficient. As long as enough markers are included, individual markers have little effect on the final metric, and even DMs calculated from mutually exclusive groups of markers correlate with each other at r~0.4-0.5. We also used data subsets to generate thousands of combinations of study populations and RPs to address sensitivity to differences in age range, sex, race, data set, sample size, and their interactions. Results were largely consistent (but not identical) regardless of the choice of RP; however, the signal was generally clearer with a younger and healthier RP, and RPs too different from the study population performed poorly. Accordingly, biomarker and RP choice are not particularly important in most cases, but caution should be used across very different populations or for fine-scale analyses. Biologically, the lack of sensitivity to marker choice and better performance of younger, healthier RPs confirm an interpretation of DM physiological dysregulation and as an emergent property of a complex system.

Many studies of aging examine biomarkers one at a time, but complex systems theory and network theory suggest that interpretations of individual markers may be context-dependent. Here, we attempted to detect underlying processes governing the levels of many biomarkers simultaneously by applying principal components analysis to 43 common clinical biomarkers measured longitudinally in 3694 humans from three longitudinal cohort studies on two continents (Women's Health and Aging I & II, InCHIANTI, and the Baltimore Longitudinal Study on Aging). The first axis was associated with anemia, inflammation, and low levels of calcium and albumin. The axis structure was precisely reproduced in all three populations and in all demographic sub-populations (by sex, race, etc.); we call the process represented by the axis "integrated albunemia." Integrated albunemia increases and accelerates with age in all populations, and predicts mortality and frailty--but not chronic disease--even after controlling for age. This suggests a role in the aging process, though causality is not yet clear. Integrated albunemia behaves more stably across populations than its component biomarkers, and thus appears to represent a higher-order physiological process emerging from the structure of underlying regulatory networks. If this is correct, detection of this process has substantial implications for physiological organization more generally.

Mechanisms maintaining honesty of sexual signals are far from resolved, limiting our understanding of sexual selection and potential important parts of physiology. Carotenoid pigmented visual signals are among the most extensively studied sexual displays, but evidence regarding hypotheses on how carotenoids ensure signal honesty is mixed. Using a phylogenetically controlled meta-analysis of 357 effect sizes across 88 different species of birds, we tested two prominent hypotheses in the field: that carotenoid-dependent coloration signals i) immunocompetence and/or ii) oxidative stress state. Separate meta-analyses were performed for the relationships of trait coloration and circulating carotenoid level with different measures of immunocompetence and oxidative stress state. For immunocompetence we find that carotenoid levels (r = 0.20) and trait color intensity (r = 0.17) are significantly positively related to PHA response. Additionally we find that carotenoids are significantly positively related to antioxidant capacity (r = 0.10), but not significantly related to oxidative damage (r = -0.02). Thus our analyses provide support for both hypotheses, in that at least for some aspects of immunity and oxidative stress state the predicted correlations were found. Furthermore, we tested for differences in effect size between experimental and observational studies; a larger effect in observational studies would indicate that co-variation might not be causal. However, we detected no significant difference, suggesting that the relationships we found are causal. The overall effect sizes we report are modest and we discuss potential factors contributing to this, including differences between species. We suggest complementary mechanisms maintaining honesty rather than the involvement of carotenoids in immune function and oxidative stress and suggest experiments on how to test these.

Understanding the dynamic relationship between components of a system or pathway at the individual cell level is a current challenge. To address this, we developed an approach that allows simultaneous tracking of several endogenous proteins of choice within individual living human cells. The approach is based on fluorescent tagging of proteins at their native locus by directed gene targeting. A fluorescent tag-encoding DNA is introduced as a new exon into the intronic region of the gene of interest, resulting in expression of a full-length fluorescently tagged protein. We used this approach to establish human cell lines simultaneously expressing two components of a major antioxidant defense system, thioredoxin 1 (Trx) and thioredoxin reductase 1 (TrxR1), labeled with CFP and YFP, respectively. We find that the distributions of both proteins between nuclear and cytoplasmic compartments were highly variable between cells. However, the two proteins did not vary independently of each other: protein levels of Trx and TrxR1 in both the whole cell and the nucleus were substantially correlated. We further find that in response to a stress-inducing drug (CPT), both Trx and TrxR1 accumulated in the nuclei in a manner that was highly temporally correlated. This accumulation considerably reduced cell-to-cell variability in nuclear content of both proteins, suggesting a uniform response of the thioredoxin system to stress. These results indicate that Trx and TrxR1 act in concert in response to stress in regard to both time course and variability. Thus, our approach provides an efficient tool for studying dynamic relationship between components of systems of interest at a single-cell level.

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human cells. As a model system, we choose three stable proteins displaying cell-cycle-dependant dynamics. We find that protein accumulation with time per cell is quadratic for proteins with long mRNA life times and approximately linear for a protein with short mRNA lifetime. Both behaviors correspond to a classical model of transcription and translation. A stochastic model, in which genes slowly switch between ON and OFF states, captures measured cell-cell variability. The data suggests, in accordance with the model, that switching to the gene ON state is exponentially distributed and that the cell-cell distribution of protein levels can be approximated by a Gamma distribution throughout the cell cycle. These results suggest that relatively simple models may describe protein dynamics in individual human cells.