Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of technologies to accomplish this has been hindered by the susceptibility of ...synthetic genetic circuits to mutation and unpredictable function during extended gut colonization. Here, we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine Escherichia coli strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
The mammalian gut is a dynamic community of symbiotic microbes that interact with the host to impact health, disease, and metabolism. We constructed engineered bacteria that survive in the mammalian ...gut and sense, remember, and report on their experiences. Based on previous genetic memory systems, we constructed a two-part system with a “trigger element” in which the lambda Cro gene is transcribed from a tetracycline-inducible promoter, and a “memory element” derived from the cI/Cro region of phage lambda. The memory element has an extremely stable cI state and a Cro state that is stable for many cell divisions. When Escherichia coli bearing the memory system are administered to mice treated with anhydrotetracycline, the recovered bacteria all have switched to the Cro state, whereas those administered to untreated mice remain in the cI state. The trigger and memory elements were transferred from E. coli K12 to a newly isolated murine E. coli strain; the stability and switching properties of the memory element were essentially identical in vitro and during passage through mice, but the engineered murine E. coli was more stably established in the mouse gut. This work lays a foundation for the use of synthetic genetic circuits as monitoring systems in complex, ill-defined environments, and may lead to the development of living diagnostics and therapeutics.
The intestine is a major source of systemic ammonia (NH
); thus, capturing part of gut NH
may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic ...encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic
Nissle 1917 to create strain SYNB1020 that converts NH
to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH
in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient
mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 10
colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma
N-nitrate (highest dose versus placebo,
= 0.0015), and urinary
N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.
To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can ...selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.
β-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis in ...several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of β-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the β-arrestin 2-targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple β-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as β-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy.
An RNA aptamer inhibits β-arrestin 2 activity.Inhibiting β-arrestin 2 impedes multiple tumorigenic pathways simultaneously.The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer.Targeting β-arrestin 2 inhibits tumor progression in CML models and patient samples.
Microbial population growth is typically measured when cells can be directly observed, or when death is rare. However, neither of these conditions hold for the mammalian gut microbiota, and, ...therefore, standard approaches cannot accurately measure the growth dynamics of this community. Here we introduce a new method (distributed cell division counting, DCDC) that uses the accurate segregation at cell division of genetically encoded fluorescent particles to measure microbial growth rates. Using DCDC, we can measure the growth rate of Escherichia coli for >10 consecutive generations. We demonstrate experimentally and theoretically that DCDC is robust to error across a wide range of temperatures and conditions, including in the mammalian gut. Furthermore, our experimental observations inform a mathematical model of the population dynamics of the gut microbiota. DCDC can enable the study of microbial growth during infection, gut dysbiosis, antibiotic therapy or other situations relevant to human health.
Density-functional theory (DFT) is used to identify phase-equilibria in multi-principal-element and high-entropy alloys (MPEAs/HEAs), including duplex-phase and eutectic microstructures. A ...combination of composition-dependent formation energy and electronic-structure-based ordering parameters were used to identify a transition from FCC to BCC favoring mixtures, and these predictions experimentally validated in the Al-Co-Cr-Cu-Fe-Ni system. A sharp crossover in lattice structure and dual-phase stability as a function of composition were predicted via DFT and validated experimentally. The impact of solidification kinetics and thermodynamic stability was explored experimentally using a range of techniques, from slow (castings) to rapid (laser remelting), which showed a decoupling of phase fraction from thermal history, i.e., phase fraction was found to be solidification rate-independent, enabling tuning of a multi-modal cell and grain size ranging from nanoscale through macroscale. Strength and ductility tradeoffs for select processing parameters were investigated via uniaxial tension and small-punch testing on specimens manufactured via powder-based additive manufacturing (directed-energy deposition). This work establishes a pathway for design and optimization of next-generation multiphase superalloys via tailoring of structural and chemical ordering in concentrated solid solutions.
Display omitted
Bacteria can be engineered to function as diagnostics or therapeutics in the mammalian gut but commercial translation of these technologies has been hindered by the susceptibility of synthetic ...genetic circuits to mutation and unpredictable function during extended gut colonization. Here we report stable, engineered bacterial strains that maintain their function for 6 months in the mouse gut. We engineered a commensal murine
Escherichia coli
strain to detect tetrathionate, which is produced during inflammation. Using our engineered diagnostic strain, which retains memory of exposure in the gut for analysis by fecal testing, we detected tetrathionate in both infection-induced and genetic mouse models of inflammation over 6 months. The synthetic genetic circuits in the engineered strain were genetically stable and functioned as intended over time. The robust, durable performance of these strains confirms the potential of engineered bacteria as living diagnostics.
To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can ...selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer- mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell-types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.
beta-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis ...in several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of beta-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the beta-arrestin 2- targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple beta-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as beta-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy. Highlights: * An RNA aptamer inhibits beta-arrestin 2 activity. * Inhibiting beta-arrestin 2 impedes multiple tumorigenic pathways simultaneously. * The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer. * Targeting beta-arrestin 2 inhibits tumor progression in CML models and patient samples.
PDF
