Misharin et al. elucidate the fate and function of monocyte-derived alveolar macrophages during the course of pulmonary fibrosis. These cells persisted throughout the life span, were enriched for the expression of profibrotic genes, and their genetic ablation ameliorated development of pulmonary fibrosis.

The release of cytokines by T cells defines a significant part of their functional activity in vivo, and their ability to produce multiple cytokines has been associated with beneficial immune responses. To date, time-integrated end-point measurements have obscured whether these polyfunctional states arise from the simultaneous or successive release of cytokines. Here, we used serial, timedependent, single-cell analysis of primary human T cells to resolve the temporal dynamics of cytokine secretion from individual cells after activation ex vivo. We show that multifunctional, Th1-skewed cytokine responses (IFN-γ, IL-2, TNFα) are initiated asynchronously, but the ensuing dynamic trajectories of these responses evolve programmatically in a sequential manner. That is, cells predominantly release one of these cytokines at a time rather than maintain active secretion of multiple cytokines simultaneously. Furthermore, these dynamic trajectories are strongly associated with the various states of cell differentiation suggesting that transient programmatic activities of many individual T cells contribute to sustained, population-level responses. The trajectories of responses by single cells may also provide unique, time-dependent signatures for immune monitoring that are less compromised by the timing and duration of integrated measures.microengraving | multifunctionality | dynamical systems | computational biology

A new, energy-based descriptor for porous materials is highly predictive for hydrogen adsorption using an interpretable regression model.

Only a tiny fraction of the nanomedicine-design space has been explored, owing to the structural complexity of nanomedicines and the lack of relevant high-throughput synthesis and analysis methods. Here, we report a methodology for determining structure–activity relationships and design rules for spherical nucleic acids (SNAs) functioning as cancer-vaccine candidates. First, we identified ~1,000 candidate SNAs on the basis of reasonable ranges for 11 design parameters that can be systematically and independently varied to optimize SNA performance. Second, we developed a high-throughput method for making SNAs at the picomolar scale in a 384-well format, and used a mass spectrometry assay to rapidly measure SNA immune activation. Third, we used machine learning to quantitatively model SNA immune activation and identify the minimum number of SNAs needed to capture optimum structure–activity relationships for a given SNA library. Our methodology is general, can reduce the number of nanoparticles that need to be tested by an order of magnitude, and could serve as a screening tool for the development of nanoparticle therapeutics.

This document contains legends for all supplementary tables and the contents for Supplementary Tables 9 and 10; the remaining supplementary tables are provided as Microsoft Excel Worksheets. Supplementary Fig. 2 Differences between spaced and compact promoters. (a) Fractional activation for dose response profiles in Fig. 2a. Fractional activation was determined by dividing each data point by the maximum reporter expression induced by the ZFa on a given reporter. In several conditions, notably ZF1x6-S, excess ZFa (above 50 ng plasmid) resulted in a decrease in reporter expression. In this case, we hypothesize that unbound ZFa competes with bound ZFa for endogenous cofactors required for transcription. (b) Representative flow cytometry histograms for experiments with spaced reporters in Fig. 2a. Data were gated on single, transfected cells ( Supplementary Fig. 15). Reporter expression increases with ZFa dose and number of binding sites. (c) Representative flow cytometry histograms from experiments with compact reporters in Fig. 2a. Data were gated on single, transfected cells. Reporter expression increases with ZFa dose and number of binding sites. Compared to the case of spaced promoters (panel b), cases with compact promoters exhibit a greater fraction of cells that are distinguishably ON, i.e., expressing more EYFP than cells without ZFa. (d) Investigating different minimal promoters with COMET. ZF1a dose responses were conducted using ZF1x6-C promoters with either the YB_TATA, CMV, or SV40 minimal promoters. The SV40 minimal promoter produced low levels of gene expression, while the YB_TATA minimal promoter conferred a maximal gene expression level similar to that of the CMV minimal promoter. Although the CMV minimal promoter was more responsive at lower levels of ZFa expression, this promoter also had higher leaky gene expression (in the absence of ZFa) than did the YB_TATA minimal promoter (quantified in Supplementary Table 10 with fitted parameters). Thus, the YB_TATA minimal promoter resulted in higher fold inductions than did the CMV minimal promoter (approximately 220-fold for YB_TATA, as compared to 60-fold for CMV_min) without sacrificing maximal gene expression. (e) Investigating maximal inducible EYFP expression. Cells were transfected with ZF1a plasmid and with reporter plasmid containing a ZF1x6-S (left) or ZF1x6-C (right) promoter. The ZFa plasmid and reporter plasmid were maintained at a ratio of 1:2 (ZFa:reporter) as the doses were scaled. On the x-axis, a value of 1 denotes a condition with 100 ng of ZF1a plasmid and 200 ng of reporter plasmid. In previous experiments, reporter expression typically plateaued at the level indicated by plasmid doses corresponding to 1 on the x-axis and could not be increased by the addition of more ZFa plasmid (Supplementary Fig. 2a). However, doubling the amount of both ZFa plasmid and reporter plasmid led to twice the reporter expression, which indicates the amount of plasmid was the limiting factor in gene expression as opposed to a downstream step such as tr...

Macrophage-initiated inflammation is tightly regulated to eliminate threats such as infections while suppressing harmful immune activation. However, individual cells' signaling responses to pro-inflammatory cues are heterogeneous, with subpopulations emerging with high or low activation states. Here, we use single-cell tracking and dynamical modeling to develop and validate a revised model for lipopolysaccharide (LPS)-induced macrophage activation that invokes a mechanism we term quorum licensing. The results show that bimodal phenotypic partitioning of macrophages is primed during the resting state, dependent on cumulative history of cell density, predicted by extrinsic noise in transcription factor expression, and independent of canonical LPS-induced intercellular feedback in the tumor necrosis factor (TNF) response. Our analysis shows how this density-dependent coupling produces a nonlinear effect on collective TNF production. We speculate that by linking macrophage density to activation, this mechanism could amplify local responses to threats and prevent false alarms.

Metabolic conditions affect the developmental tempo of most animal species. Consequently, developmental gene regulatory networks (GRNs) must faithfully adjust their dynamics to a variable time scale. We find evidence that layered weak repression of genes provides the necessary coupling between GRN output and cellular metabolism. Using a mathematical model that replicates such a scenario, we find that lowering metabolism corrects developmental errors that otherwise occur when different layers of repression are lost. Through mutant analysis, we show that gene expression dynamics are unaffected by loss of repressors, but only when cellular metabolism is reduced. We further show that when metabolism is lowered, formation of a variety of sensory organs in Drosophila is normal despite loss of individual repressors of transcription, mRNA stability, and protein stability. We demonstrate the universality of this phenomenon by experimentally eliminating the entire microRNA family of repressors, and find that all microRNAs are rendered unnecessary when metabolism is reduced. Thus, layered weak repression provides robustness through error frequency suppression, and may provide an evolutionary route to a shorter reproductive cycle. ! 2. CC-BY-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The circadian clock, which coordinates daily physiological behaviors of most organisms, maintains endogenous (approximately 24 h) cycles and simultaneously synchronizes to the 24-h environment due to its inherent robustness to environmental perturbations coupled with a sensitivity to specific environmental stimuli. In this study, the authors develop a detailed mathematical model that characterizes the Drosophila melanogaster circadian network. This model incorporates the transcriptional regulation of period, time-less, vrille, PAR-domain protein 1, and clock gene and protein counterparts. The interlocked positive and negative feedback loops that arise from these clock components are described primarily through mass-action kinetics (with the exception of regulated gene expression) and without the use of explicit time delays. System parameters are estimated via a genetic algorithm-based optimization of a cost function that relies specifically on circadian phase behavior since amplitude measurements are often noisy and do not account for the unique entrainment features that define circadian oscillations. Resulting simulations of this 29-state ordinary differential equation model comply with fitted wild-type experimental data, demonstrating accurate free-running (23.24-h periodic) and entrained (24-h periodic) circadian dynamics. This model also predicts unfitted mutant phenotype behavior by illustrating short and long periodicity, robust oscillations, and arrhythmicity. This mechanistic model also predicts light-induced circadian phase resetting (as described by the phase-response curve) that are in line with experimental observations.