Mechano-regulation of Wnt/ß-Catenin signaling to control paraxial versus lateral mesoderm lineage bifurcation.

Mechanical cues are involved in many biological processes, including embryonic development and patterning. For example, external mechanical forces (shear stress), lateral cell-cell interactions, and mechanical properties (stiffness and composition) of the extracellular matrix are thought to modulate Wnt signaling, which is a highly conserved pathway involved in regulating stem cell renewal, proliferation, and differentiation. In this work, we employed a customized higher-throughput shear stress induction device for the controlled application of mechanical stress to study the effects of shear stress on the differentiation of human induced pluripotent stem cells (hiPSCs) toward the three germ layers. We found that mechanical stress alters lineage commitment during ectoderm and mesoderm differentiation. We show that this effect correlates with reduced Wnt signaling, evaluated in terms of the promoter activity of an established TCF3-responsive promoter. Whole transcriptome sequencing and pathway enrichment analysis of the differentially expressed genes between hiPSC-derived mesoderm cells differentiated in the presence or absence of piston-induced shear stress confirmed that Wnt/ß-catenin signaling is among the most affected developmental pathways. Furthermore, our results suggest that suitably programmed shear stress application could be used to selectively promote differentiation of hiPSCs to either lateral or paraxial mesoderm in commercially available media.

Programmable DARPin-based receptors for the detection of thrombotic markers.

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

Control of gene expression in engineered mammalian cells with a programmable shear-stress inducer.

In humans, cellular mechanoperception serves as the basis of touch sensation and proprioception, contributes to the proper programming of cell fate during embryonic development, and plays a pivotal role in the development of mechanosensitive tissues. Molecular mechanoreceptors can respond to their environment by mediating transient adjustments of ion homeostasis, which subsequently trigger calcium-dependent alteration of gene expression via specific signaling pathways such as the nuclear factor of the activated T-cells pathway. Although, mechanoreceptors are potential drug targets for various diseases, current techniques to study mechanically gated processes are often based on custom-tailored microfluidic systems, which require special setups or have limited throughput. Here, we present a platform to characterize shear-stress-triggered, calcium-mediated gene expression, which employs a programmable, 96-well-format, shear-stress induction device to examine the effects of imposing various mechanical loads on mammalian adherent cell lines. The presented method is suitable for high-throughput experiments and provides a large tunable parameter space to optimize conditions for different cell types. Our findings indicate that the device is an effective tool to explore conditions in terms of frequency, intensity, intervals as well as extracellular matrix composition alongside the evaluation of different combinations of mechanosensitive proteins for mechanically activated gene expression. We believe our results can serve as a platform for further investigations into shear stress-controlled gene expression in basic research and drug screening.

Smartphone-Flashlight-Mediated Remote Control of Rapid Insulin Secretion Restores Glucose Homeostasis in Experimental Type-1 Diabetes.

Emerging digital assessment of biomarkers by linking health-related data obtained from wearable electronic devices and embedded health and fitness sensors in smartphones is opening up the possibility of creating a continuous remote-monitoring platform for disease management. It is considered that the built-in flashlight of smartphones may be utilized to remotely program genetically engineered designer cells for on-demand delivery of protein-based therapeutics. Here, the authors present smartphone-induced insulin release in ß-cell line (iß-cell) technology for traceless light-triggered rapid insulin secretion, employing the light-activatable receptor melanopsin to induce calcium influx and membrane depolarization upon illumination. This iß-cell-based system enables repeated, reversible secretion of insulin within 15 min in response to light stimulation, with a high induction fold both in vitro and in vivo. It is shown that programmable percutaneous remote control of implanted microencapsulated iß-cells with a smartphone's flashlight rapidly reverses hyperglycemia in a mouse model of type-1 diabetes.

Smart-watch-programmed green-light-operated percutaneous control of therapeutic transgenes.

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.

A versatile plasmid architecture for mammalian synthetic biology (VAMSyB).

Current molecular cloning strategies generally lack inter-compatibility, are not strictly modular, or are not applicable to engineer multi-gene expression vectors for transient and stable integration. A standardized molecular cloning platform would advance research, for example, by promoting exchange of vectors between groups. Here, we present a versatile plasmid architecture for mammalian synthetic biology, which we designate VAMSyB, consisting of a three-tier vector family. Tier-1 is designed for easy engineering of fusion constructs, as well as easy swapping of genes and modules to tune the functionality of the vector. Tier-2 is designed for transient multi-gene expression, and is constructed by directly transferring the engineered expression cassettes from tier-1 vectors. Tier-3 enables stable integration into a mammalian host cell through viral transduction, transposons, or homology-directed recombination via CRISPR. This VAMSyB architecture is expected to have broad applicability in the field of mammalian synthetic biology. The VAMSyB collection of plasmids will be available through Addgene.

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.

Construction of a Multiwell Light-Induction Platform for Traceless Control of Gene Expression in Mammalian Cells.

Mammalian cells can be engineered to incorporate light-responsive elements that reliably sense stimulation by light and activate endogenous pathways, such as the cAMP or Ca2+ pathway, to control gene expression. Light-inducible gene expression systems offer high spatiotemporal resolution, and are also traceless, reversible, tunable, and inexpensive. Melanopsin, a well-known representative of the animal opsins, is a G-protein-coupled receptor that triggers a Gαq-dependent signaling cascade upon activation with blue light (≈470 nm). Here, we describe how to rewire melanopsin activation by blue light to transgene expression in mammalian cells, with detailed instructions for constructing a 96-LED array platform with multiple tunable parameters for illumination of the engineered cells in multiwell plates.

Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells.

We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.

Neurons differentiate magnitude and location of mechanical stimuli.

Neuronal activity can be modulated by mechanical stimuli. To study this phenomenon quantitatively, we mechanically stimulated rat cortical neurons by shear stress and local indentation. Neurons show 2 distinct responses, classified as transient and sustained. Transient responses display fast kinetics, similar to spontaneous neuronal activity, whereas sustained responses last several minutes before returning to baseline. Local soma stimulations with micrometer-sized beads evoke transient responses at low forces of ∼220 nN and pressures of ∼5.6 kPa and sustained responses at higher forces of ∼360 nN and pressures of ∼9.2 kPa. Among the neuronal compartments, axons are highly susceptible to mechanical stimulation and predominantly show sustained responses, whereas the less susceptible dendrites predominantly respond transiently. Chemical perturbation experiments suggest that mechanically evoked responses require the influx of extracellular calcium through ion channels. We propose that subtraumatic forces/pressures applied to neurons evoke neuronal responses via nonspecific gating of ion channels.

Light-Controlled Mammalian Cells and Their Therapeutic Applications in Synthetic Biology.

The ability to remote control the expression of therapeutic genes in mammalian cells in order to treat disease is a central goal of synthetic biology-inspired therapeutic strategies. Furthermore, optogenetics, a combination of light and genetic sciences, provides an unprecedented ability to use light for precise control of various cellular activities with high spatiotemporal resolution. Recent work to combine optogenetics and therapeutic synthetic biology has led to the engineering of light-controllable designer cells, whose behavior can be regulated precisely and noninvasively. This Review focuses mainly on non-neural optogenetic systems, which are often used in synthetic biology, and their applications in genetic programing of mammalian cells. Here, a brief overview of the optogenetic tool kit that is available to build light-sensitive mammalian cells is provided. Then, recently developed strategies for the control of designer cells with specific biological functions are summarized. Recent translational applications of optogenetically engineered cells are also highlighted, ranging from in vitro basic research to in vivo light-controlled gene therapy. Finally, current bottlenecks, possible solutions, and future prospects for optogenetics in synthetic biology are discussed.

Generalized extracellular molecule sensor platform for programming cellular behavior.

Strategies for expanding the sensor space of designer receptors are urgently needed to tailor cell-based therapies to respond to any type of medically relevant molecules. Here, we describe a universal approach to designing receptor scaffolds that enables antibody-specific molecular input to activate JAK/STAT, MAPK, PLCG or PI3K/Akt signaling rewired to transgene expression driven by synthetic promoters. To demonstrate its scope, we equipped the GEMS (generalized extracellular molecule sensor) platform with antibody fragments targeting a synthetic azo dye, nicotine, a peptide tag and the PSA (prostate-specific antigen) biomarker, thereby covering inputs ranging from small molecules to proteins. These four GEMS devices provided robust signaling and transgene expression with high signal-to-noise ratios in response to their specific ligands. The sensitivity of the nicotine- and PSA-specific GEMS devices matched the clinically relevant concentration ranges, and PSA-specific GEMS were able to detect pathological PSA levels in the serum of patients diagnosed with prostate cancer.

Preferential Extracellular Generation of the Active Parkinsonian Toxin MPP+ by Transporter-Independent Export of the Intermediate MPDP+.

AIMS: 1-Methyl-4-phenyl-tetrahydropyridine (MPTP) is among the most widely used neurotoxins for inducing experimental parkinsonism. MPTP causes parkinsonian symptoms in mice, primates, and humans by killing a subpopulation of dopaminergic neurons. Extrapolations of data obtained using MPTP-based parkinsonism models to human disease are common; however, the precise mechanism by which MPTP is converted into its active neurotoxic metabolite, 1-methyl-4-phenyl-pyridinium (MPP(+)), has not been fully elucidated. In this study, we aimed to address two unanswered questions related to MPTP toxicology: (1) Why are MPTP-converting astrocytes largely spared from toxicity? (2) How does MPP(+) reach the extracellular space? RESULTS: In MPTP-treated astrocytes, we discovered that the membrane-impermeable MPP(+), which is generally assumed to be formed inside astrocytes, is almost exclusively detected outside of these cells. Instead of a transporter-mediated export, we found that the intermediate, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP(+)), and/or its uncharged conjugate base passively diffused across cell membranes and that MPP(+) was formed predominately by the extracellular oxidation of MPDP(+) into MPP(+). This nonenzymatic extracellular conversion of MPDP(+) was promoted by O2, a more alkaline pH, and dopamine autoxidation products. INNOVATION AND CONCLUSION: Our data indicate that MPTP metabolism is compartmentalized between intracellular and extracellular environments, explain the absence of toxicity in MPTP-converting astrocytes, and provide a rationale for the preferential formation of MPP(+) in the extracellular space. The mechanism of transporter-independent extracellular MPP(+) formation described here indicates that extracellular genesis of MPP(+) from MPDP is a necessary prerequisite for the selective uptake of this toxin by catecholaminergic neurons.

A method for multiplex gene synthesis employing error correction based on expression.

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.

Synthesis and biological evaluation of optimized inhibitors of the mitotic kinesin Kif18A.

The mitotic spindle, a highly dynamic structure composed of microtubules, mediates the segregation of the previously duplicated genome into the two nascent daughter cells. Errors in this process contribute to pathology including tumor formation. Key for the shape and function of the mitotic spindle are kinesins, molecular motor proteins that convert chemical energy into mechanical work. Due to their fast mode of action, small molecules are valuable tools to dissect the dynamic functions of kinesins during mitosis. In this study, we report the identification of optimized small molecule inhibitors of the mitotic kinesin Kif18A. Using BTB-1, the first identified Kif18A inhibitor, as a lead compound, we synthesized a collection of derivatives. We demonstrate that some of the synthesized derivatives potently inhibited the ATPase activity of Kif18A with a half maximal inhibitory concentration (IC50) value in the low micromolar range. In vitro analysis of a panel of Kif18A-related kinesins revealed that the two most potent compounds show improved selectivity compared to BTB-1. Structure-activity relationship studies identified substituents mediating undesired inhibitory effects on microtubule polymerization. In summary, our study provides key insights into the mechanism of action of BTB-1 and its analogs, which will have a great impact on the further development of highly selective and bioactive Kif18A inhibitors. Since Kif18A is frequently overexpressed in solid tumors, such compounds are not only of great interest for basic research but also have the potential to open up new strategies for the treatment of human diseases.

Structure-function relationship of thiazolide-induced apoptosis in colorectal tumor cells.

Thiazolides are a novel class of anti-infectious agents against intestinal intracellular and extracellular protozoan parasites, bacteria, and viruses. While the parent compound nitazoxanide (NTZ; 2-(acetolyloxy)-N-(5-nitro-2-thiazolyl)benzamide) has potent antimicrobial activity, the bromo-thiazolide RM4819 (N-(5-bromothiazol-2-yl)-2-hydroxy-3-methylbenzamide) shows only reduced activity. Interestingly, both molecules are able to induce cell death in colon carcinoma cell lines, indicating that the molecular target in intestinal pathogens and in colon cancer cells is different. The detoxification enzyme glutathione S-transferase of class Pi 1 (GSTP1) is frequently overexpressed in various tumors, including colon carcinomas, and limits the efficacy of antitumor chemotherapeutic drugs due to its detoxifying activities. In colorectal tumor cells RM4819 has been shown to interact with GSTP1, and GSTP1 enzymatic activity is required for thiazolide-induced apoptosis. At present it is unclear which molecular structures of RM4819 are required to interact with GSTP1 and to induce cell death in colon carcinoma cell lines. Here, we demonstrate that novel thiazolide derivatives with variation in their substituents of the benzene ring do not significantly affect apoptosis induction in Caco-2 cells, whereas removal of the bromide atom on the thiazole ring leads to a strong reduction of cell death induction in colon cancer cells. We further show that active thiazolides require caspase activation and GSTP1 expression in order to induce apoptosis. We demonstrate that increased glutathione (GSH) levels sensitize colon cancer cells to thiazolides, indicating that both GSTP1 enzymatic activity as well as GSH levels are critical factors in thiazolide-induced cell death.

Thiamine pyrophosphate stimulates acetone activation by Desulfococcus biacutus as monitored by a fluorogenic ATP analogue.

Acetone can be degraded by aerobic and anaerobic microorganisms. Studies with the strictly anaerobic sulfate-reducing bacterium Desulfococcus biacutus indicate that acetone degradation by these bacteria starts with an ATP-dependent carbonylation reaction leading to acetoacetaldehyde as the first reaction product. The reaction represents the second example of a carbonylation reaction in the biochemistry of strictly anaerobic bacteria, but the exact mechanism and dependence on cofactors are still unclear. Here, we use a novel fluorogenic ATP analogue to investigate its mechanism. We find that thiamine pyrophosphate is a cofactor of this ATP-dependent reaction. The products of ATP cleavage are AMP and pyrophosphate, providing first insights into the reaction mechanism by indicating that the reaction proceeds without intermediate formation of acetone enol phosphate.

Expanding the scope of human DNA polymerase λ and ß inhibitors.

The exact biological functions of individual DNA polymerases still await clarification, and therefore appropriate reagents to probe their respective functions are required. In the present study, we report the development of a highly potent series of human DNA polymerase λ and ß (pol λ and ß) inhibitors based on the rhodanine scaffold. Both enzymes are involved in DNA repair and are thus considered as future drug targets. We expanded the chemical diversity of the small-molecule inhibitors arising from a high content screening and designed and synthesized 30 novel analogues. By biochemical evaluation, we discovered 23 highly active compounds against pol λ. Importantly, 10 of these small-molecules selectively inhibited pol λ and not the homologous pol ß. We discovered 14 small-molecules that target pol ß and found out that they are more potent than known inhibitors. We also investigated whether the discovered compounds sensitize cancer cells toward DNA-damaging reagents. Thus, we cotreated human colorectal cancer cells (Caco-2) with the small-molecule inhibitors and hydrogen peroxide or the approved drug temozolomide. Interestingly, the tested compounds sensitized Caco-2 cells to both genotoxic agents in a DNA repair pathway-dependent manner.

Small Molecule Inhibitors of Human DNA Polymerase λ.

To discover chemical probes to further under-stand the function of individual DNA polymerases, we established a generally applicable high-throughput screening. By applying this technique we discovered three novel inhibitor classes of human DNA polymerase λ (DNA Pol λ), a key enzyme to maintain the genetic integrity of the genome. The rhodanines, classified as an excellent drug scaffold, were found to be the most potent inhibitors for DNA Pol λ. Importantly, they are up to 10 times less active against the highly similar DNA polymerase ß. We investigated basic structure activity relationships. Furthermore, the rhodanines showed pharmacological activity in two human cancer cell lines. So the here reported small molecules could serve as useful DNA Pol λ probes and might serve as starting point to develop novel therapeutic agents.