Synthetic mammalian signaling circuits for robust cell population control.

In multicellular organisms, cells actively sense and control their own population density. Synthetic mammalian quorum-sensing circuits could provide insight into principles of population control and extend cell therapies. However, a key challenge is reducing their inherent sensitivity to "cheater" mutations that evade control. Here, we repurposed the plant hormone auxin to enable orthogonal mammalian cell-cell communication and quorum sensing. We designed a paradoxical population control circuit, termed "Paradaux," in which auxin stimulates and inhibits net cell growth at different concentrations. This circuit limited population size over extended timescales of up to 42 days of continuous culture. By contrast, when operating in a non-paradoxical regime, population control became more susceptible to mutational escape. These results establish auxin as a versatile "private" communication system and demonstrate that paradoxical circuit architectures can provide robust population control.

Substrate stress relaxation regulates monolayer fluidity and leader cell formation for collectively migrating epithelia.

Collective migration of epithelial tissues is a critical feature of developmental morphogenesis and tissue homeostasis. Coherent motion of cell collectives requires large scale coordination of motion and force generation and is influenced by mechanical properties of the underlying substrate. While tissue viscoelasticity is a ubiquitous feature of biological tissues, its role in mediating collective cell migration is unclear. Here, we have investigated the impact of substrate stress relaxation on the migration of micropatterned epithelial monolayers. Epithelial monolayers exhibit faster collective migration on viscoelastic alginate substrates with slower relaxation timescales, which are more elastic, relative to substrates with faster stress relaxation, which exhibit more viscous loss. Faster migration on slow-relaxing substrates is associated with reduced substrate deformation, greater monolayer fluidity, and enhanced leader cell formation. In contrast, monolayers on fast-relaxing substrates generate substantial substrate deformations and are more jammed within the bulk, with reduced formation of transient lamellipodial protrusions past the monolayer edge leading to slower overall expansion. This work reveals features of collective epithelial dynamics on soft, viscoelastic materials and adds to our understanding of cell-substrate interactions at the tissue scale. Significance Statement: Groups of cells must coordinate their movements in order to sculpt organs during development and maintain tissues. The mechanical properties of the underlying substrate on which cells reside are known to influence key aspects of single and collective cell migration. Despite being a nearly universal feature of biological tissues, the role of viscoelasticity (i.e., fluid-like and solid-like behavior) in collective cell migration is unclear. Using tunable engineered biomaterials, we demonstrate that sheets of epithelial cells display enhanced migration on slower-relaxing (more elastic) substrates relative to faster-relaxing (more viscous) substrates. Building our understanding of tissue-substrate interactions and collective cell dynamics provides insights into approaches for tissue engineering and regenerative medicine, and therapeutic interventions to promote health and treat disease.

Simultaneous removal of ammonia nitrogen, sulfamethoxazole, and antibiotic resistance genes in self-corrosion microelectrolysis-enhanced counter-diffusion biofilm system.

A self-corrosion microelectrolysis (SME)-enhanced membrane-aerated biofilm reactor (eMABR) was developed for the removal of pollutants and reduction of antibiotic resistance genes (ARGs). Fe2+ and Fe3+ formed iron oxides on the biofilm, which enhanced the adsorption and redox process. SME can induce microorganisms to secrete more extracellular proteins and up-regulate the expression of ammonia monooxygenase (AMO) (0.92 log2). AMO exposed extra binding sites (ASP-69) for antibiotics, weakening the competition between NH4+-N and sulfamethoxazole (SMX). The NH4+-N removal efficiency in the S-eMABR (adding SMX and IC) increased by 44.87 % compared to the S-MABR (adding SMX). SME increased the removal performance of SMX by approximately 1.45 times, down-regulated the expressions of sul1 (-1.69 log2) and sul2 (-1.30 log2) genes, and controlled their transfer within the genus. This study provides a novel strategy for synergistic reduction of antibiotics and ARGs, and elucidates the corresponding mechanism based on metatranscriptomic and molecular docking analyses.

Tuning Methylation-Dependent Silencing Dynamics by Synthetic Modulation of CpG Density.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here, we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.

Tuning methylation-dependent silencing dynamics by synthetic modulation of CpG density.

Methylation of cytosines in CG dinucleotides (CpGs) within promoters has been shown to lead to gene silencing in mammals in natural contexts. Recently, engineered recruitment of methyltransferases (DNMTs) at specific loci was shown to be sufficient to silence synthetic and endogenous gene expression through this mechanism. A critical parameter for DNA methylation-based silencing is the distribution of CpGs within the target promoter. However, how the number or density of CpGs in the target promoter affects the dynamics of silencing by DNMT recruitment has remained unclear. Here we constructed a library of promoters with systematically varying CpG content, and analyzed the rate of silencing in response to recruitment of DNMT. We observed a tight correlation between silencing rate and CpG content. Further, methylation-specific analysis revealed a constant accumulation rate of methylation at the promoter after DNMT recruitment. We identified a single CpG site between TATA box and transcription start site (TSS) that accounted for a substantial part of the difference in silencing rates between promoters with differing CpG content, indicating that certain residues play disproportionate roles in controlling silencing. Together, these results provide a library of promoters for synthetic epigenetic and gene regulation applications, as well as insights into the regulatory link between CpG content and silencing rate.