Towards an Electronic Health Prevention Record Based on HL7 FHIR and the OMOP Common Data Model.

BACKGROUND: Approximately 40% of all recorded deaths in Austria are due to behavioral risks. These risks could be avoided with appropriate measures. OBJECTIVES: Extension of the concept of EHR and EMR to an electronic prevention record, focusing on primary and secondary prevention. METHODS: The concept of a structured prevention pathway, based on the principles of P4 Medicine, was developed for a multidisciplinary prevention network. An IT infrastructure based on HL7 FHIR and the OHDSI OMOP common data model was designed. RESULTS: An IT solution supporting a structured and modular prevention pathway was conceptualized. It contained a personalized management of prevention, risk assessment, diagnostic and preventive measures supported by a modular, interoperable IT infrastructure including a health app, prevention record web-service, decision support modules and a smart prevention registry, separating primary and secondary use of data. CONCLUSION: A concept was created on how an electronic health prevention record based on HL7 FHIR and the OMOP common data model can be implemented.

Design of a Biohybrid Materials Circuit with Binary Decoder Functionality.

Synthetic biology applies concepts from electrical engineering and information processing to endow cells with computational functionality. Transferring the underlying molecular components into materials and wiring them according to topologies inspired by electronic circuit boards has yielded materials systems that perform selected computational operations. However, the limited functionality of available building blocks is restricting the implementation of advanced information-processing circuits into materials. Here, a set of protease-based biohybrid modules the bioactivity of which can either be induced or inhibited is engineered. Guided by a quantitative mathematical model and following a design-build-test-learn (DBTL) cycle, the modules are wired according to circuit topologies inspired by electronic signal decoders, a fundamental motif in information processing. A 2-input/4-output binary decoder for the detection of two small molecules in a material framework that can perform regulated outputs in form of distinct protease activities is designed. The here demonstrated smart material system is strongly modular and can be used for biomolecular information processing for example in advanced biosensing or drug delivery applications.

Reversible Shielding and Immobilization of Liposomes and Viral Vectors by Tailored Antibody-Ligand Interactions.

Controlling the time and dose of nanoparticulate drug delivery by administration of small molecule drugs holds promise for efficient and safer therapies. This study describes a versatile approach of exploiting antibody-ligand interactions for the design of small molecule-responsive nanocarrier and nanocomposite systems. For this purpose, antibody fragments (scFvs) specific for two distinct small molecule ligands are designed. Subsequently, the surface of nanoparticles (liposomes or adeno-associated viral vectors, AAVs) is modified with these ligands, serving as anchor points for scFv binding. By modifying the scFvs with polymer tails, they can act as a non-covalently bound shielding layer, which is recruited to the anchor points on the nanoparticle surface and prevents interactions with cultured mammalian cells. Administration of an excess of the respective ligand triggers competitive displacement of the shielding layer from the nanoparticle surface and restores nanoparticle-cell interactions. The same principle is applied for developing hydrogel depots that can release integrated AAVs or liposomes in response to small molecule ligands. The liberated nanoparticles subsequently deliver their cargoes to cells. In summary, the utilization of different antibody-ligand interactions, different nanoparticles, and different release systems validates the versatility of the design concept described herein.

Biosensor-Enabled Multiplexed On-Site Therapeutic Drug Monitoring of Antibiotics.

Personalized antibiotherapy ensures that the antibiotic concentration remains in the optimal therapeutic window to maximize efficacy, minimize side effects, and avoid the emergence of drug resistance due to insufficient dosing. However, such individualized schemes need frequent sampling to tailor the blood antibiotic concentrations. To optimally integrate therapeutic drug monitoring (TDM) into the clinical workflow, antibiotic levels can either be measured in blood using point-of-care testing (POCT), or can rely on noninvasive sampling. Here, a versatile biosensor with an antibody-free assay for on-site TDM is presented. The platform is evaluated with an animal study, where antibiotic concentrations are quantified in different matrices including whole blood, plasma, urine, saliva, and exhaled breath condensate (EBC). The clearance and the temporal evaluation of antibiotic levels in EBC and plasma are demonstrated. Influence of matrix effects on measured drug concentrations is determined by comparing the plasma levels with those in noninvasive samples. The system's potential for blood-based POCT is further illustrated by tracking ß-lactam concentrations in untreated blood samples. Finally, multiplexing capabilities are explored successfully for multianalyte/sample analysis. By enabling a rapid, low-cost, sample-independent, and multiplexed on-site TDM, this system can shift the paradigm of "one-size-fits-all" strategy.

Spatiotemporally confined red light-controlled gene delivery at single-cell resolution using adeno-associated viral vectors.

Methodologies for the controlled delivery of genetic information into target cells are of utmost importance for genetic engineering in both fundamental and applied research. However, available methods for efficient gene transfer into user-selected or even single cells suffer from low throughput, the need for complicated equipment, high invasiveness, or side effects by off-target viral uptake. Here, we engineer an adeno-associated viral (AAV) vector system that transfers genetic information into native target cells upon illumination with cell-compatible red light. This OptoAAV system allows adjustable and spatially resolved gene transfer down to single-cell resolution and is compatible with different cell lines and primary cells. Moreover, the sequential application of multiple OptoAAVs enables spatially resolved transduction with different transgenes. The approach presented is likely extendable to other classes of viral vectors and is expected to foster advances in basic and applied genetic research.

Synthetic Biology: Emerging Concepts to Design and Advance Adeno-Associated Viral Vectors for Gene Therapy.

Three recent approvals and over 100 ongoing clinical trials make adeno-associated virus (AAV)-based vectors the leading gene delivery vehicles in gene therapy. Pharmaceutical companies are investing in this small and nonpathogenic gene shuttle to increase the therapeutic portfolios within the coming years. This prospect of marking a new era in gene therapy has fostered both investigations of the fundamental AAV biology as well as engineering studies to enhance delivery vehicles. Driven by the high clinical potential, a new generation of synthetic-biologically engineered AAV vectors is on the rise. Concepts from synthetic biology enable the control and fine-tuning of vector function at different stages of cellular transduction and gene expression. It is anticipated that the emerging field of synthetic-biologically engineered AAV vectors can shape future gene therapeutic approaches and thus the design of tomorrow's gene delivery vectors. This review describes and discusses the recent trends in capsid and vector genome engineering, with particular emphasis on synthetic-biological approaches.

Synthetic Biology-Empowered Hydrogels for Medical Diagnostics.

Synthetic biology is strongly inspired by concepts of engineering science and aims at the design and generation of artificial biological systems in different fields of research such as diagnostics, analytics, biomedicine, or chemistry. To this aim, synthetic biology uses an engineering approach relying on a toolbox of molecular sensors and switches that endows cellular hosts with non-natural computing functions and circuits. Importantly, this concept is not only limited to cellular approaches. Synthetic biological building blocks have also conferred sensing and switching capability to otherwise inactive materials. This principle has attracted high interest for the development of biohybrid materials capable of sensing and responding to specific molecular stimuli, such as disease biomarkers, antibiotics, or heavy metals. Moreover, the interconnection of individual sense-and-respond materials to complex materials systems has enabled the processing of, for example, multiple inputs or the amplification of signals using feedback topologies. Such systems holding high potential for applications in the analytical and diagnostic sectors will be described in this chapter.

Structural Characterization and Lifetimes of Triple-Stranded Helical Coinage Metal Complexes: Synthesis, Spectroscopy and Quantum Chemical Calculations.

This work reports on a series of polynuclear complexes containing a trinuclear Cu, Ag, or Au core in combination with the fac-isomer of the metalloligand [Ru(pypzH)3 ](PF6 )2 (pypzH=3-(pyridin-2-yl)pyrazole). These (in case of the Ag and Au containing species) newly synthesized compounds of the general formula [{Ru(pypz)3 }2 M3 ](PF6 ) (2: M=Cu; 3: M=Ag; 4: M=Au) contain triple-stranded helical structures in which two ruthenium moieties are connected by three N-M-N (M=Cu, Ag, Au) bridges. In order to obtain a detailed description of the structure both in the electronic ground and excited states, extensive spectroscopic and quantum chemical calculations are applied. The equilateral coinage metal core triangle in the electronic ground state of 2-4 is distorted in the triplet state. Furthermore, the analyses offer a detailed description of electronic excitations. By using time-resolved IR spectroscopy from the microsecond down to the nanosecond regime, both the vibrational spectra and the lifetime of the lowest lying electronically excited triplet state can be determined. The lifetimes of these almost only non-radiative triplet states of 2-4 show an unusual effect in a way that the Au-containing complex 4 has a lifetime which is by more than a factor of five longer than in case of the Cu complex 2. Thus, the coinage metals have a significant effect on the electronically excited state, which is localized on a pypz ligand coordinated to the Ru atom indicating an unusual cooperative effect between two moieties of the complex.

Phosphine-functionalised tris(pyrazolyl)methane ligands and their mono- and heterobimetallic complexes.

The synthesis, characterisation and reactivity of new phosphine-functionalised tris(pyrazolyl)methane ligands (TpmPR2, 2a-c, with R = Ph, nBu, iPr) are presented. The reaction of 2a-c with [Rh(CO)2Cl]2 furnished N,P-heterochelate carbonyl complexes (3a-c), which were used to quantify the donor abilities of the ligands via IR spectroscopy. The coordination flexibility was demonstrated by treating representative members of this new ligand class with [CpRu(acn)3][PF6] (acn = acetonitrile), [(tht)AuCl] (tht = tetrahydrothiophene) and [Pd(allyl)Cl]2 providing either a N,N,P-heteroscorpionate complex (4) or the P-coordinated complexes (5,6) without any involvement of the pyrazolyl entities. With 2a and [Pd(cod)Cl2], another P,N-heterochelate complex (7) was obtained, which served as a precursor for a heterobimetallic complex containing palladium and copper (8). Detailed NMR spectroscopic and X-ray crystallographic investigations have been performed on all new complexes.

Design of a Human Rhinovirus-14 3C Protease-Inducible Caspase-3.

The engineering of enzymes for the purpose of controlling their activity represents a valuable approach to address challenges in both fundamental and applied research. Here, we describe and compare different design strategies for the generation of a human rhinovirus-14 (HRV14) 3C protease-inducible caspase-3 (CASP3). We exemplify the application potential of the resulting protease by controlling the activity of a synthetic enzyme cascade, which represents an important motif for the design of artificial signal transduction networks. In addition, we use our engineered CASP3 to characterize the effect of aspartate mutations on enzymatic activity. Besides the identification of mutations that render the enzyme inactive, we find the CASP3-D192E mutant (aspartate-to-glutamate exchange at position 192) to be inaccessible for 3C protease-mediated cleavage. This indicates a structural change of CASP3 that goes beyond a slight misalignment of the catalytic triad. This study could inspire the design of additional engineered proteases that could be used to unravel fundamental research questions or to expand the collection of biological parts for the design of synthetic signaling pathways.

Biofunctionalized Materials Featuring Feedforward and Feedback Circuits Exemplified by the Detection of Botulinum Toxin A.

Feedforward and feedback loops are key regulatory elements in cellular signaling and information processing. Synthetic biology exploits these elements for the design of molecular circuits that enable the reprogramming and control of specific cellular functions. These circuits serve as a basis for the engineering of complex cellular networks, opening the door for numerous medical and biotechnological applications. Here, a similar principle is applied. Feedforward and positive feedback circuits are incorporated into biohybrid polymer materials in order to develop signal-sensing and signal-processing devices. This concept is exemplified by the detection of the proteolytic activity of the botulinum neurotoxin A. To this aim, site-specific proteases are incorporated into receiver, transmitter, and output materials, and their release, diffusion, and/or activation are wired according to a feedforward or a positive feedback circuit. The development of a quantitative mathematical model enables analysis and comparison of the performance of both systems. The flexible design could be easily adapted to detect other toxins or molecules of interest. Furthermore, cellular signaling or gene regulatory pathways could provide additional blueprints for the development of novel biohybrid circuits. Such information-processing, material-embedded biological circuits hold great promise for a variety of analytical, medical, or biotechnological applications.

Redox-responsive phosphonite gold complexes in hydroamination catalysis.

Very high activities were observed in the redox-induced hydroamination of alkynes by employing a redox-active gold(i) complex featuring an electron-deficient, terphenyl-substituted phosphonite-based ligand. The hydroamination proceeds roughly two-fold faster with the in situ oxidized catalysts than with their reduced form.

A Two-Step Approach for the Design and Generation of Nanobodies.

Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

Characterization of the synthetic biology-inspired implementation of a materials-based positive feedback loop.

The translation of engineering designs to materials sciences by means of synthetic biological tools represents a novel concept for the development of information-processing materials systems. Here, we provide data on the mathematical model-guided implementation of a biomaterials-based positive feedback loop for the detection of proteolytic activities. Furthermore, we present data on an extended system design for the detection of the antibiotic novobiocin. This work is related to the research article "Synthetic biology-inspired design of signal-amplifying materials systems" (Wagner et al., 2018) [1].

Engineering bacterial microcompartments with heterologous enzyme cargos.

Bacterial microcompartments (BMCs) are intracellular proteinaceous organelles devoid of a lipid membrane that encapsulates enzymes of metabolic pathways. Salmonella enterica synthesizes propanediol-utilization BMCs containing enzymes involved in the degradation of 1,2-propanediol. BMCs can be designed to enclose heterologous proteins, paving the way to engineered catalytic microreactors. Here, we investigate broader applicability of this design principle by directing three different enzymes to the BMC. We demonstrate that ß-galactosidase, esterase Est5, and cofactor-dependent glycerol dehydrogenase can be directed to the BMC and copurified with the microcompartment shell in a catalytically active form. We show that the BMC shell protects enzymes from pH-dependent but not from temperature stress. Moreover, we provide evidence that the heterologously expressed BMCs act as a moderately selective diffusion barrier for lipophilic small molecules.

Upgrading biomaterials with synthetic biological modules for advanced medical applications.

One key aspect of synthetic biology is the development and characterization of modular biological building blocks that can be assembled to construct integrated cell-based circuits performing computational functions. Likewise, the idea of extracting biological modules from the cellular context has led to the development of in vitro operating systems. This principle has attracted substantial interest to extend the repertoire of functional materials by connecting them with modules derived from synthetic biology. In this respect, synthetic biological switches and sensors, as well as biological targeting or structure modules, have been employed to upgrade functions of polymers and solid inorganic material. The resulting systems hold great promise for a variety of applications in diagnosis, tissue engineering, and drug delivery. This review reflects on the most recent developments and critically discusses challenges concerning in vivo functionality and tolerance that must be addressed to allow the future translation of such synthetic biology-upgraded materials from the bench to the bedside.

A Boron-Fluorinated Tris(pyrazolyl)borate Ligand ((F) Tp*) and Its Mono- and Dinuclear Copper Complexes [Cu((F) Tp*)2 ] and [Cu2 ((F) Tp*)2 ]: Synthesis, Structures, and DFT Calculations.

Reaction of [Si(3,5-Me2 pz)4 ] (1) with [Cu(MeCN)4 ][BF4 ] (2) gave the mono- and dinuclear copper complexes [Cu2 ((F) Tp*)2 ] (3) and [Cu((F) Tp*)2 ] (4). Both complexes contain the so-far unprecedented boron-fluorinated (F) Tp* ligand ([FB(3,5-Me2 pz)3 ](-) with pz=pyrazolyl) originating from 1, acting as a pyrazolyl transfer reagent, and the [BF4 ](-) counter anion of 2, serving as the source of the {BF} entity. The solid-state structures as well as the NMR and EPR spectroscopic characteristics of the complexes were elaborated. Pulsed gradient spin echo (PGSE) experiments revealed that 3 retains (almost entirely) its dimeric structure in benzene, whereas dimer cleavage and formation of acetonitrile adducts, presumably [Cu((F) Tp*)(MeCN)], is observed in acetonitrile. The short Cu⋅⋅⋅Cu distance of 269.16 pm in the solid-state is predicted by DFT calculations to be dictated by dispersion interactions between all atoms in the complex (the Cu-Cu dispersion contribution itself is only very small). As revealed by cyclic voltammetry studies, 3 shows an irreversible (almost quasi-reversible at higher scan rates) oxidation process centred at E(pa) =-0.23 V (E(0) 1/2 =-0.27 V) (vs. Fc/Fc(+) ). Oxidation reactions on a preparative scale with one equivalent of the ferrocenium salt [Fc][BF4 ] (very slow reaction) or air (fast reaction) furnished blue crystals of the mononuclear copper(II) complex [Cu((F) Tp*)2 ] (4). As expected for a Jahn-Teller-active system, the coordination sphere around copper(II) is strongly distorted towards a stretched octahedron, in accordance with EPR spectroscopic findings.

Optogenetically controlled RAF to characterize BRAF and CRAF protein kinase inhibitors.

Here, we applied optoRAF, an optogenetic tool for light-controlled clustering and activation of RAF proteins that mimics the natural occurring RAS-mediated dimerization. This versatile tool allows studying the effect on BRAF and CRAF homodimer- as well as heterodimer-induced RAF signaling. Vemurafenib and dabrafenib are two clinically approved inhibitors for BRAF that efficiently suppress the kinase activity of oncogenic BRAF (V600E). However in wild-type BRAF expressing cells, BRAF inhibitors can exert paradoxical activation of wild-type CRAF. Using optoRAF, vemurafenib was identified as paradoxical activator of BRAF and CRAF homo- and heterodimers. Dabrafenib enhanced activity of light-stimulated CRAF at low dose and inhibited CRAF signaling at high dose. Moreover, dabrafenib increased the protein level of CRAF proteins but not of BRAF proteins. Increased CRAF levels correlate with elevated RAF signaling in a dabrafenib-dependent manner, independent of light activation.

Modularized CRISPR/dCas9 effector toolkit for target-specific gene regulation.

The ability to control mammalian genes in a synergistic mode using synthetic transcription factors is highly desirable in fields of tissue engineering, stem cell reprogramming and fundamental research. In this study, we developed a standardized toolkit utilizing an engineered CRISPR/Cas9 system that enables customizable gene regulation in mammalian cells. The RNA-guided dCas9 protein was implemented as a programmable transcriptional activator or repressor device, including targeting of endogenous loci. For facile assembly of single or multiple CRISPR RNAs, our toolkit comprises a modular RNAimer plasmid, which encodes the required noncoding RNA components.

Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy.

The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT).