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.

Antagonistic control of a dual-input mammalian gene switch by food additives.

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.

Synthetic mammalian trigger-controlled bipartite transcription factors.

Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), γ-butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and γ-butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.

Enhancing the safety of CAR-T cell therapy: Synthetic genetic switch for spatiotemporal control.

Chimeric antigen receptor T (CAR-T) cell therapy is a promising and precise targeted therapy for cancer that has demonstrated notable potential in clinical applications. However, severe adverse effects limit the clinical application of this therapy and are mainly caused by uncontrollable activation of CAR-T cells, including excessive immune response activation due to unregulated CAR-T cell action time, as well as toxicity resulting from improper spatial localization. Therefore, to enhance controllability and safety, a control module for CAR-T cells is proposed. Synthetic biology based on genetic engineering techniques is being used to construct artificial cells or organisms for specific purposes. This approach has been explored in recent years as a means of achieving controllability in CAR-T cell therapy. In this review, we summarize the recent advances in synthetic biology methods used to address the major adverse effects of CAR-T cell therapy in both the temporal and spatial dimensions.

A review of the degradation of antibiotic contaminants using advanced oxidation processes: modification and application of layered double hydroxides based materials.

In recent years, the treatment of organic pollutants has become a global concern due to the threat to human health posed by emerging contaminants, especially antibiotic contamination. Advanced oxidation processes (AOPs) can solve the organic pollution problem well, which have been identified as a promising solution for the treatment of hard-to-handle organic compounds including antibiotic contaminants. Layered double hydroxides (LDHs) are excellent catalysts because of their flexible tunability, favorable thermal stability, abundant active sites, and facile exchangeability of intercalated anions. This paper conducted a systematic review of LDHs-based materials used for common antibiotic removal by three significant AOP technologies, such as photocatalysis, the Fenton-like processes, and peroxymonosulfate catalysis. The degradation effects studied in various studies were reviewed, and the mechanisms were discussed in detail based on the type of AOPs. Finally, the challenges and the application trends of AOPs that may arise were prospected. The aim of this study is to suggest ways to provide practical guidance for the screening and improvement of LDH materials and the rational selection of AOPs to achieve efficient antibiotic degradation. This could lead to the development of more efficient and environmentally friendly materials and processes for degrading antibiotics, with significant implications for our ecological conservation by addressing water pollution.

Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells.

Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.

Identification of Sclareol As a Natural Neuroprotective Cav 1.3-Antagonist Using Synthetic Parkinson-Mimetic Gene Circuits and Computer-Aided Drug Discovery.

Parkinson's disease (PD) results from selective loss of substantia nigra dopaminergic (SNc DA) neurons, and is primarily caused by excessive activity-related Ca2+ oscillations. Although L-type voltage-gated calcium channel blockers (CCBs) selectively inhibiting Cav 1.3 are considered promising candidates for PD treatment, drug discovery is hampered by the lack of high-throughput screening technologies permitting isoform-specific assessment of Cav-antagonistic activities. Here, a synthetic-biology-inspired drug-discovery platform enables identification of PD-relevant drug candidates. By deflecting Cav-dependent activation of nuclear factor of activated T-cells (NFAT)-signaling to repression of reporter gene translation, they engineered a cell-based assay where reporter gene expression is activated by putative CCBs. By using this platform in combination with in silico virtual screening and a trained deep-learning neural network, sclareol is identified from a essential oils library as a structurally distinctive compound that can be used for PD pharmacotherapy. In vitro studies, biochemical assays and whole-cell patch-clamp recordings confirmed that sclareol inhibits Cav 1.3 more strongly than Cav 1.2 and decreases firing responses of SNc DA neurons. In a mouse model of PD, sclareol treatment reduced DA neuronal loss and protected striatal network dynamics as well as motor performance. Thus, sclareol appears to be a promising drug candidate for neuroprotection in PD patients.

Enhancing mechanism of arsenic(iii) adsorption by MnO2-loaded calcined MgFe layered double hydroxide.

The use of MnO2/MgFe-layered double hydroxide (MnO2/MgFe-LDH) and MnO2/MgFe-layered double oxide (MnO2/MgFe-LDO400 °C) for arsenic immobilization from the aqueous medium is the subject of this research. Fourier transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy were used to characterise MnO2/MgFe-LDH and MnO2/MgFe-LDO400 °C. Based on our developed method, MnO2 was spread on the clay composites' surfaces in the form of a chemical bond. The clay composite exhibited a good adsorption effect on arsenic. The experimental findings fit the pseudo-second-order model well, indicating that the chemisorption mechanism played a significant role in the adsorption process. Furthermore, the Freundlich model suited the adsorption isotherm data of all adsorbents well. The recycling experiment showed that MnO2/MgFe-LDH and MnO2/MgFe-LDO400 °C exhibited good stability and reusability. In summary, MnO2/MgFe-LDH and MnO2/MgFe-LDO400 °C are promising for developing processes for efficient control of the pollutant arsenic.

Engineering precision therapies: lessons and motivations from the clinic.

In the past decade, gene- and cell-based therapies have been at the forefront of the biomedical revolution. Synthetic biology, the engineering discipline of building sophisticated 'genetic software' to enable precise regulation of gene activities in living cells, has been a decisive success factor of these new therapies. Here, we discuss the core technologies and treatment strategies that have already gained approval for therapeutic applications in humans. We also review promising preclinical work that could either enhance the efficacy of existing treatment strategies or pave the way for new precision medicines to treat currently intractable human conditions.

Engineering Mammalian Cells to Control Glucose Homeostasis.

Diabetes mellitus is a complex metabolic disease characterized by chronically deregulated blood-glucose levels. To restore glucose homeostasis, therapeutic strategies allowing well-controlled production and release of insulinogenic hormones into the blood circulation are required. In this chapter, we describe how mammalian cells can be engineered for applications in diabetes treatment. While closed-loop control systems provide automated and self-sufficient synchronization of glucose sensing and drug production, drug production in open-loop control systems is engineered to depend on exogenous user-defined trigger signals. Rational, robust, and reliable manufacture practices for mammalian cell engineering are essential for industrial-scale mass-production in view of clinical and commercial applications.

miR-371b-5p promotes cell proliferation, migration and invasion in non-small cell lung cancer via SCAI.

OBJECTIVE: Multiple gene targets have been reported for treatment of non-small cell lung cancer (NSCLC), however, the accompanying genetic tolerance was reported increasingly. Therefore, it is important to find new biomarkers or therapeutic targets in treatment of NSCLC. METHODS: The expression levels of miR-371b-5p were detected by qRT-PCR in NSCLC tissues and cell lines. To evaluate the effect of miR-371b-5p on NSCLC progression, we first transfected the miR-371b-5p inhibitor for construction of the miR-371b-5p down-regulated cell model. Then the cell proliferation, migration, invasion and cell apoptosis were detected. In addition, the expression levels of adhesion factors were detected. The target gene of miR-371b-5p was identified by bioinformatics analysis, and rescue experiment was conducted to validate the effect of miR-371b-5p on proliferation, migration and invasion of NSCLC. RESULTS: Our findings revealed that the miR-371b-5p was overexpressed in NSCLC and could markedly promote the cell proliferation, migration and invasion. Expression levels of both intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were significantly down-regulated when treated by miR-371b-5p inhibitor. Moreover, dual-luciferase reporter assay showed that the miR-371b-5p targeted SCAI in regulation of cell proliferation, migration and invasion, and the expression of miR-371b-5p was negatively associated with SCAI in NSCLC tissues and cell lines. Rescue experiment revealed that the miR-371b-5p could rescue the effect of SCAI on cell proliferation, migration and invasion. CONCLUSION: Our results suggest that the miR-371b-5p and SCAI may serve as novel prognostic biomarkers and therapeutic targets for NSCLC.

Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice.

Sophisticated devices for remote-controlled medical interventions require an electrogenetic interface that uses digital electronic input to directly program cellular behavior. We present a cofactor-free bioelectronic interface that directly links wireless-powered electrical stimulation of human cells to either synthetic promoter-driven transgene expression or rapid secretion of constitutively expressed protein therapeutics from vesicular stores. Electrogenetic control was achieved by coupling ectopic expression of the L-type voltage-gated channel CaV1.2 and the inwardly rectifying potassium channel Kir2.1 to the desired output through endogenous calcium signaling. Focusing on type 1 diabetes, we engineered electrosensitive human ß cells (Electroß cells). Wireless electrical stimulation of Electroß cells inside a custom-built bioelectronic device provided real-time control of vesicular insulin release; insulin levels peaked within 10 minutes. When subcutaneously implanted, this electrotriggered vesicular release system restored normoglycemia in type 1 diabetic mice.