Multi-layered computational gene networks by engineered tristate logics.

So far, biocomputation strictly follows traditional design principles of digital electronics, which could reach their limits when assembling gene circuits of higher complexity. Here, by creating genetic variants of tristate buffers instead of using conventional logic gates as basic signal processing units, we introduce a tristate-based logic synthesis (TriLoS) framework for resource-efficient design of multi-layered gene networks capable of performing complex Boolean calculus within single-cell populations. This sets the stage for simple, modular, and low-interference mapping of various arithmetic logics of interest and an effectively enlarged engineering space within single cells. We not only construct computational gene networks running full adder and full subtractor operations at a cellular level but also describe a treatment paradigm building on programmable cell-based therapeutics, allowing for adjustable and disease-specific drug secretion logics in vivo. This work could foster the evolution of modern biocomputers to progress toward unexplored applications in precision medicine.

Immunomimetic Designer Cells Protect Mice from MRSA Infection.

Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.

Designing cell function: assembly of synthetic gene circuits for cell biology applications.

Synthetic biology is the discipline of engineering application-driven biological functionalities that were not evolved by nature. Early breakthroughs of cell engineering, which were based on ectopic (over)expression of single sets of transgenes, have already had a revolutionary impact on the biotechnology industry, regenerative medicine and blood transfusion therapies. Now, we require larger-scale, rationally assembled genetic circuits engineered to programme and control various human cell functions with high spatiotemporal precision in order to solve more complex problems in applied life sciences, biomedicine and environmental sciences. This will open new possibilities for employing synthetic biology to advance personalized medicine by converting cells into living therapeutics to combat hitherto intractable diseases.

Peroxymonosulfate activation by CuMn-LDH for the degradation of bisphenol A: Effect, mechanism, and pathway.

The remediation of water contaminated with bisphenol A (BPA) has gained significant attention. In this study, a hydrothermal composite activator of Cu3Mn-LDH containing coexisting phases of cupric nitrate (Cu(NO3)2) and manganous nitrate (Mn(NO3)2) was synthesized. Advanced oxidation processes were employed as an effective approach for BPA degradation, utilizing Cu3Mn-LDH as the catalyst to activate peroxymonosulfate (PMS). The synthesis of the Cu3Mn-LDH material was characterized using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). According to the characterization data and screening experiments, Cu3Mn-LDH was selected as the best experimental material. Cu3Mn-LDH exhibits remarkable catalytic ability with PMS, demonstrating good degradation efficiency of BPA under neutral and alkaline conditions. With a PMS dosage of 0.25 g·L-1 and Cu3Mn-LDH dosage of 0.10 g·L-1, 10 mg·L-1 BPA (approximately 17.5 µM) can be completely degraded within 40 min, of which the TOC removal reached 95%. The reactive oxygen species present in the reaction system were analyzed by quenching experiments and EPR. Results showed that sulfate free radicals (SO4•-), hydroxyl free radicals (•OH), superoxide free radicals (•O2-), and nonfree radical mono-oxygen were generated, while mono-oxygen played a key role in degrading BPA. Cu3Mn-LDH exhibits excellent reproducibility, as it can still completely degrade BPA even after four consecutive cycles. The degradation intermediates of BPA were detected by GCMS, and the possible degradation pathways were reasonably predicted. This experiment proposes a nonradical degradation mechanism for BPA and analyzes the degradation pathways. It provides a new perspective for the treatment of organic pollutants in water.

Efficient Adsorption Capacity of MgFe-Layered Double Hydroxide Loaded on Pomelo Peel Biochar for Cd (II) from Aqueous Solutions: Adsorption Behaviour and Mechanism.

A novel pomelo peel biochar/MgFe-layered double hydroxide composite (PPBC/MgFe-LDH) was synthesised using a facile coprecipitation approach and applied to remove cadmium ions (Cd (II)). The adsorption isotherm demonstrated that the Cd (II) adsorption by the PPBC/MgFe-LDH composite fit the Langmuir model well, and the adsorption behaviour was a monolayer chemisorption. The maximum adsorption capacity of Cd (II) was determined to be 448.961 (±12.3) mg·g-1 from the Langmuir model, which was close to the actual experimental adsorption capacity 448.302 (±1.41) mg·g-1. The results also demonstrated that the chemical adsorption controlled the rate of reaction in the Cd (II) adsorption process of PPBC/MgFe-LDH. Piecewise fitting of the intra-particle diffusion model revealed multi-linearity during the adsorption process. Through associative characterization analysis, the adsorption mechanism of Cd (II) of PPBC/MgFe-LDH involved (i) hydroxide formation or carbonate precipitation; (ii) an isomorphic substitution of Fe (III) by Cd (II); (iii) surface complexation of Cd (II) by functional groups (-OH); and (iv) electrostatic attraction. The PPBC/MgFe-LDH composite demonstrated great potential for removing Cd (II) from wastewater, with the advantages of facile synthesis and excellent adsorption capacity.

Gene switch for l-glucose-induced biopharmaceutical production in mammalian cells.

In this study, we designed and built a gene switch that employs metabolically inert l-glucose to regulate transgene expression in mammalian cells via d-idonate-mediated control of the bacterial regulator LgnR. To this end, we engineered a metabolic cascade in mammalian cells to produce the inducer molecule d-idonate from its precursor l-glucose by ectopically expressing the Paracoccus species 43P-derived catabolic enzymes LgdA, LgnH, and LgnI. To obtain ON- and OFF-switches, we fused LgnR to the human transcriptional silencer domain Krüppel associated box (KRAB) and the viral trans-activator domain VP16, respectively. Thus, these artificial transcription factors KRAB-LgnR or VP16-LgnR modulated cognate promoters containing LgnR-specific binding sites in a d-idonate-dependent manner as a direct result of l-glucose metabolism. In a proof-of-concept experiment, we show that the switches can control production of the model biopharmaceutical rituximab in both transiently and stably transfected HEK-293T cells, as well as CHO-K1 cells. Rituximab production reached 5.9 µg/ml in stably transfected HEK-293T cells and 3.3 µg/ml in stably transfected CHO-K1 cells.

Segregated Nanocompartments Containing Therapeutic Enzymes and Imaging Compounds within DNA-Zipped Polymersome Clusters for Advanced Nanotheranostic Platform.

Nanotheranostics is an emerging field that brings together nanoscale-engineered materials with biological systems providing a combination of therapeutic and diagnostic strategies. However, current theranostic nanoplatforms have serious limitations, mainly due to a mismatch between the physical properties of the selected nanomaterials and their functionalization ease, loading ability, or overall compatibility with bioactive molecules. Herein, a nanotheranostic system is proposed based on nanocompartment clusters composed of two different polymersomes linked together by DNA. Careful design and procedure optimization result in clusters segregating the therapeutic enzyme human Dopa decarboxylase (DDC) and fluorescent probes for the detection unit in distinct but colocalized nanocompartments. The diagnostic compartment provides a twofold function: trackability via dye loading as the imaging component and the ability to attach the cluster construct to the surface of cells. The therapeutic compartment, loaded with active DDC, triggers the cellular expression of a secreted reporter enzyme via production of dopamine and activation of dopaminergic receptors implicated in atherosclerosis. This two-compartment nanotheranostic platform is expected to provide the basis of a new treatment strategy for atherosclerosis, to expand versatility and diversify the types of utilizable active molecules, and thus by extension expand the breadth of attainable applications.

A Synthetic-Biology-Inspired Therapeutic Strategy for Targeting and Treating Hepatogenous Diabetes.

Hepatogenous diabetes is a complex disease that is typified by the simultaneous presence of type 2 diabetes and many forms of liver disease. The chief pathogenic determinant in this pathophysiological network is insulin resistance (IR), an asymptomatic disease state in which impaired insulin signaling in target tissues initiates a variety of organ dysfunctions. However, pharmacotherapies targeting IR remain limited and are generally inapplicable for liver disease patients. Oleanolic acid (OA) is a plant-derived triterpenoid that is frequently used in Chinese medicine as a safe but slow-acting treatment in many liver disorders. Here, we utilized the congruent pharmacological activities of OA and glucagon-like-peptide 1 (GLP-1) in relieving IR and improving liver and pancreas functions and used a synthetic-biology-inspired design principle to engineer a therapeutic gene circuit that enables a concerted action of both drugs. In particular, OA-triggered short human GLP-1 (shGLP-1) expression in hepatogenous diabetic mice rapidly and simultaneously attenuated many disease-specific metabolic failures, whereas OA or shGLP-1 monotherapy failed to achieve corresponding therapeutic effects. Collectively, this work shows that rationally engineered synthetic gene circuits are capable of treating multifactorial diseases in a synergistic manner by multiplexing the targeting efficacies of single therapeutics.

Cosmetics-triggered percutaneous remote control of transgene expression in mice.

Synthetic biology has significantly advanced the rational design of trigger-inducible gene switches that program cellular behavior in a reliable and predictable manner. Capitalizing on genetic componentry, including the repressor PmeR and its cognate operator OPmeR, that has evolved in Pseudomonas syringae pathovar tomato DC3000 to sense and resist plant-defence metabolites of the paraben class, we have designed a set of inducible and repressible mammalian transcription-control devices that could dose-dependently fine-tune transgene expression in mammalian cells and mice in response to paraben derivatives. With an over 60-years track record as licensed preservatives in the cosmetics industry, paraben derivatives have become a commonplace ingredient of most skin-care products including shower gels, cleansing toners and hand creams. As parabens can rapidly reach the bloodstream of mice following topical application, we used this feature to percutaneously program transgene expression of subcutaneous designer cell implants using off-the-shelf commercial paraben-containing skin-care cosmetics. The combination of non-invasive, transdermal and orthogonal trigger-inducible remote control of transgene expression may provide novel opportunities for dynamic interventions in future gene and cell-based therapies.

A synthetic biology-based device prevents liver injury in mice.

BACKGROUND & AIMS: The liver performs a panoply of complex activities coordinating metabolic, immunologic and detoxification processes. Despite the liver's robustness and unique self-regeneration capacity, viral infection, autoimmune disorders, fatty liver disease, alcohol abuse and drug-induced hepatotoxicity contribute to the increasing prevalence of liver failure. Liver injuries impair the clearance of bile acids from the hepatic portal vein which leads to their spill over into the peripheral circulation where they activate the G-protein-coupled bile acid receptor TGR5 to initiate a variety of hepatoprotective processes. METHODS: By functionally linking activation of ectopically expressed TGR5 to an artificial promoter controlling transcription of the hepatocyte growth factor (HGF), we created a closed-loop synthetic signalling network that coordinated liver injury-associated serum bile acid levels to expression of HGF in a self-sufficient, reversible and dose-dependent manner. RESULTS: After implantation of genetically engineered human cells inside auto-vascularizing, immunoprotective and clinically validated alginate-poly-(L-lysine)-alginate beads into mice, the liver-protection device detected pathologic serum bile acid levels and produced therapeutic HGF levels that protected the animals from acute drug-induced liver failure. CONCLUSIONS: Genetically engineered cells containing theranostic gene circuits that dynamically interface with host metabolism may provide novel opportunities for preventive, acute and chronic healthcare. LAY SUMMARY: Liver diseases leading to organ failure may go unnoticed as they do not trigger any symptoms or significant discomfort. We have designed a synthetic gene circuit that senses excessive bile acid levels associated with liver injuries and automatically produces a therapeutic protein in response. When integrated into mammalian cells and implanted into mice, the circuit detects the onset of liver injuries and coordinates the production of a protein pharmaceutical which prevents liver damage.