Orthogonal Control Research Articles

Orthogonal programming of heterogeneous micro-mechano-environments and geometries in three-dimensional bio-stereolithography

Engineering heterogeneous micro-mechano-microenvironments of extracellular matrix is of great interest in tissue engineering, but spatial control over mechanical heterogeneity in three dimensions is still challenging given the fact that geometry and stiffness are inherently intertwined in fabrication. Here, we develop a layer-by-layer three-dimensional (3D) printing paradigm which achieves orthogonal control of stiffness and geometry by capitalizing on the conventionally adverse effect of oxygen inhibition on free-radical polymerization. Controlled oxygen permeation and inhibition result in photo-cured hydrogel layers with thicknesses only weakly dependent to the ultraviolet exposure dosage. The dosage is instead leveraged to program the crosslink density and stiffness of the cured structures. The programmable stiffness spans nearly an order of magnitude (E ~ 2–15 kPa) within the physiologically relevant range. We further demonstrate that extracellular matrices with programmed micro-mechano-environments can dictate 3D cellular organization, enabling in vitro tissue reconstruction.

Elucidating the Kinetic and Thermodynamic Driving Forces in Polymer Blend Film Self-Stratification

Self-assembly of immiscible polymer blend films during deposition presents an attractive avenue to fabricate structured multicomponent thin films. Depth profiles formed by self-stratification during spin-casting can be varied simply by modifying processing conditions, even on short time scales. However, the current practice of self-stratification utilizes polymer blends that are specific for optimizing properties of a desired application and provides little insight into the underlying driving forces that guide the process of stratification. Our research seeks to fill this gap by using neutron reflectivity to monitor the stratification of blends consisting of poly(3-hexylthiophene-2,5-diyl) (P3HT) and poly(methyl methacrylate) (PMMA) as a function of sample and processing condition: spin-casting speed, polymer blend composition, and polymer molecular weight. Each of these parameters was varied individually, and each parameter provided orthogonal control of the extent of stratification. An increase in the c...

Pop-Up Conducting Large-Area Biographene Kirigami.

We demonstrate the rapid, large-area transformation of bioenabled graphene laminates into multidimensional geometries for pop-up and stretchable applications. Water-vapor annealing facilitates the controlled plasticization of the multilayered silk-graphene morphologies, allowing highly localized kirigami cuts by programmable drag knife with diverse type and depth of cuts. By adjusting drag-knife depth, we can generate a microscale array of full and partial cuts, enabling a purely topological approach toward the control of metastable fold-unfold states and crack fracture paths in kirigami structures. Through orthogonal control over the graphene-silk composite's nanoscale morphology, cut pattern, and semimetal-like conductivity, we showcase bioenabled laminates as a platform for prospective soft and shape-transforming electronics as flexible interconnects and stretchable energy harvesters.

Mechanically tunable conductive interpenetrating network hydrogels that mimic the elastic moduli of biological tissue

Conductive and stretchable materials that match the elastic moduli of biological tissue (0.5–500 kPa) are desired for enhanced interfacial and mechanical stability. Compared with inorganic and dry polymeric conductors, hydrogels made with conducting polymers are promising soft electrode materials due to their high water content. Nevertheless, most conducting polymer-based hydrogels sacrifice electronic performance to obtain useful mechanical properties. Here we report a method that overcomes this limitation using two interpenetrating hydrogel networks, one of which is formed by the gelation of the conducting polymer PEDOT:PSS. Due to the connectivity of the PEDOT:PSS network, conductivities up to 23 S m−1 are achieved, a record for stretchable PEDOT:PSS-based hydrogels. Meanwhile, the low concentration of PEDOT:PSS enables orthogonal control over the composite mechanical properties using a secondary polymer network. We demonstrate tunability of the elastic modulus over three biologically relevant orders of magnitude without compromising stretchability ( > 100%) or conductivity ( > 10 S m−1).

Linkage-Specific Detection and Metabolism of Human Milk Oligosaccharides in Escherichia coli

Human milk oligosaccharides (HMOs) are important prebiotic complex carbohydrates with demonstrated beneficial effects on the microbiota of neonates. However, optimization of their biotechnological synthesis is limited by the relatively low throughput of monosaccharide and linkage analysis. To enable high-throughput screening of HMO structures, we constructed a whole-cell biosensor that uses heterologous expression of glycosidases to generate linkage-specific, quantitative fluorescent readout for a range of HMOs at detection limits down to 20μM in approximately 6hr. We also demonstrate the use of this system for orthogonal control of growth rate or protein expression of particular strains in mixed populations. This work enables rapid non-chromatographic linkage analysis and lays the groundwork for the application of directed evolution to biosynthesis of complex carbohydrates as well as the prebiotic manipulation of population dynamics in natural and engineered microbial communities.

Current inverting metamutator, its implementation with a new single active device and applications

A new kind of metamutator, namely "Current Inverting Metamutator", its realizations using different types of active blocks and some of its applications like voltage-mode universal biquadratic filter with three input and one output terminals are presented. The proposed circuits can realize all standard filters, namely, low-pass, band-pass, high-pass, notch and all-pass without passive component matching conditions. The proposed circuit offers the features of using grounded capacitors and orthogonal controllability of angular frequency and quality factor. Then a novel realization of metamutator with one active device, additive and differential IC (AD-IC) is proposed and implemented with twelve transistors only. The metamutator with AD-IC has the advantages: (1) of creating new realizations of memristors, capacitance multipliers, inductor simulators, frequency dependent negative resistors which can be used to make IC active filters, (2) less is the number (only one) of active devices, less is the amount of disparity, (3) no need to match passive component values.

A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Timing of DNA damage responses impacts persistence to fluoroquinolones

Bacterial persisters are subpopulations of phenotypic variants in isogenic cultures that can survive lethal doses of antibiotics. Their tolerances are often attributed to reduced activities of antibiotic targets, which limit corruption and damage in persisters compared with bacteria that die from treatment. However, that model does not hold for nongrowing populations treated with ofloxacin, a fluoroquinolone, where antibiotic-induced damage is comparable between cells that live and those that die. To understand how those persisters achieve this feat, we employed a genetic system that uses orthogonal control of MazF and MazE, a toxin and its cognate antitoxin, to generate model persisters that are uniformly tolerant to ofloxacin. Despite this complete tolerance, MazF model persisters required the same DNA repair machinery (RecA, RecB, and SOS induction) to survive ofloxacin treatment as their nongrowing, WT counterparts and exhibited similar indicators of DNA damage from treatment. Further investigation revealed that, following treatment, the timing of DNA repair was critical to MazF persister survival because, when repair was delayed until after growth and DNA synthesis resumed, survival was compromised. In addition, we found that, with nongrowing, WT planktonic and biofilm populations, stalling the resumption of growth and DNA synthesis after the conclusion of fluoroquinolone treatment with a prevalent type of stress at infection sites (nutrient limitation) led to near complete survival. These findings illustrate that the timing of events, such as DNA repair, following fluoroquinolone treatment is important to persister survival and provide further evidence that knowledge of the postantibiotic recovery period is critical to understanding persistence phenotypes.

Independent Control over Multiple Cell Types in Space and Time Using Orthogonal Blue and Red Light Switchable Cell Interactions.

Independent control over multiple cell–material interactions with high spatiotemporal resolution is a key for many biomedical applications and understanding cell biology, as different cell types can perform different tasks in a multicellular context. In this study, the binding of two different cell types to materials is orthogonally controlled with blue and red light providing independent regulation in space and time. Cells expressing the photoswitchable protein cryptochrome 2 (CRY2) on cell surface bind to N‐truncated CRY‐interacting basic helix–loop–helix protein 1 (CIBN)‐immobilized substrates under blue light and cells expressing the photoswitchable protein phytochrome B (PhyB ) on cell surface bind to phytochrome interaction factor 6 (PIF6)‐immobilized substrates under red light, respectively. These light‐switchable cell interactions provide orthogonal and noninvasive control using two wavelengths of visible light. Moreover, both cell–material interactions are dynamically switched on under light and reversible in the dark. The specificity of the CRY2/CIBN and PhyB/PIF6 interactions and their response to different wavelengths of light allow selectively activating the binding of one cell type with blue and the other cell type with red light in the presence of the other cell type.

Synthetic Biology of Small RNAs and Riboswitches.

In bacteria and archaea, small RNAs (sRNAs) regulate complex networks through antisense interactions with target mRNAs in trans, and riboswitches regulate gene expression in cis based on the ability to bind small-molecule ligands. Although our understanding and characterization of these two important regulatory RNA classes is far from complete, these RNA-based mechanisms have proven useful for a wide variety of synthetic biology applications. Besides classic and contemporary applications in the realm of metabolic engineering and orthogonal gene control, this review also covers newer applications of regulatory RNAs as biosensors, logic gates, and tools to determine RNA-RNA interactions. A separate section focuses on critical insights gained and challenges posed by fundamental studies of sRNAs and riboswitches that should aid future development of synthetic regulatory RNAs.

Human Cell Encapsulation in Gel Microbeads with Cosynthesized Concentric Nanoporous Solid Shells

AbstractEncapsulation of therapeutic cells in core–shell microparticles has great promise for the treatment of a range of health conditions. Unresolved challenges related to control of the particle morphology, mechanical stability, and immunogenicity hinder dissemination of this promising approach. Here, a novel polymer material for cell encapsulation and a combined novel, easy to control, synthesis method are introduced. Core–shell cell encapsulation is demonstrated with a concentric core–shell morphology formed during a single UV exposure, resulting in particles that consist of a synthetic hydrogel core of polyethylene glycol diacrylate and a solid, but porous, shell of off‐stoichiometric thiol‐ene. The encapsulated human cells in 100 µm diameter particles have >90% viability. The average shell thickness is controlled between 7 and 13 µm by varying the UV exposure, and the shell is measured to be permeable to low molecular weight species (<180 Da) but impermeable to higher molecular weight species (>480 Da). The unique material properties and the orthogonal control of the microparticle core size, shell thickness, shell permeability, and shell surface properties address the key unresolved challenges in the field, and are expected to enable faster translation of novel cell therapy concepts from research to clinical practice.

Remote Electronic Control of DNA-Based Reactions and Nanostructure Assembly.

The use of synthetic DNA to design and build molecular machines and well-defined structures at the nanoscale has greatly impacted the field of nanotechnology. Here we expand the current toolkit in this field by demonstrating an efficient, quantitative, and versatile approach that allows us to remotely control DNA-based reactions and DNA nanostructure self-assembly using electronic inputs. To do so we have deposited onto the surface of disposable chips different DNA input strands that upon the application of a cathodic potential can be desorbed in a remote and controlled way and trigger DNA-based reactions and DNA nanostructure self-assembly. We demonstrate that this effect is specific and versatile and allows the orthogonal control of multiple reactions and multiple structures in the same solution. Moreover, the strategy is highly tunable and can be finely modulated by varying the cathodic potential, the period of applied potential, and the density of the DNA strand on the chip surface. Our approach thus represents a versatile way to remotely control DNA-based circuits and nanostructure assembly and can allow new possible applications of DNA-based nanotools.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Chemical Control of a CRISPR-Cas9 Acetyltransferase.

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Toward this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First, we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tunable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

Energy-efficient adjustable speed double inverter-fed woundrotor induction motor drive

The mathematical and analytical models of adjustable speed electric drive based on double inverter-fed induction motor are considered and analyzed in the article, the main equations which describe energy performances of such type of AC electric drive system are determined. The influence of excitation frequency on energy performances of double inverter-fed induction motor in steady-state at orthogonal flux vector control is considered in the article. The condition ensures minimum of stator steel losses at a predetermined rotor speed and can operate at speeds up to double rated without the magnetic flux reducing is presented. This possibility is one of the most important advantages of this type of electric drive over others types of AC drives for many technological processes.

High-Efficiency mosfet-Based MMC Design for LVDC Distribution Systems

Low-voltage dc (LVdc) distribution networks have the potential to release larger capacity without having to upgrade the existing cables. One of the main challenges of LVdc networks is the extra customer-end dc–ac conversion stage. This paper proposes and evaluates a five-level Si mosfet -based modular multilevel converter (MMC) as a promising alternative to the conventional two-level insulated gate bipolar transistor-based converter. This is due to the comparatively higher efficiency, power quality and reliability, and reduced electromagnetic (EM) emissions. A comprehensive analysis of a Si mosfet five-level MMC converter design is performed to investigate the suitability of the topology for LVdc applications. Detailed theoretical analysis of the five-level MMC is presented, with simulated and experimental results to demonstrate circuit performance. To suppress the ac circulating current, especially the dominant second harmonics, this paper presents a double line-frequency proportional integral (PI) with orthogonal imaginary axis control method. Comparison of simulation and experimental results with those for double line-frequency proportional resonant control shows that the proposed PI controller has a better performance. In addition, it is simpler to implement and more immune to sampling/discretization errors.

Fundamental Design Principles for Transcription-Factor-Based Metabolite Biosensors.

Metabolite biosensors are central to current efforts toward precision engineering of metabolism. Although most research has focused on building new biosensors, their tunability remains poorly understood and is fundamental for their broad applicability. Here we asked how genetic modifications shape the dose-response curve of biosensors based on metabolite-responsive transcription factors. Using the lac system in Escherichia coli as a model system, we built promoter libraries with variable operator sites that reveal interdependencies between biosensor dynamic range and response threshold. We developed a phenomenological theory to quantify such design constraints in biosensors with various architectures and tunable parameters. Our theory reveals a maximal achievable dynamic range and exposes tunable parameters for orthogonal control of dynamic range and response threshold. Our work sheds light on fundamental limits of synthetic biology designs and provides quantitative guidelines for biosensor design in applications such as dynamic pathway control, strain optimization, and real-time monitoring of metabolism.

Engineered extracellular matrices with controlled mechanics modulate renal proximal tubular cell epithelialization.

Acute kidney injury (AKI) is common and associated with significant morbidity and mortality. Recovery from many forms of AKI involves the proliferation of renal proximal tubular epithelial cells (RPTECs), but the influence of the microenvironment in which this recovery occurs remains poorly understood. Here we report the development of a poly(ethylene glycol) (PEG) hydrogel platform to study the influence of substrate mechanical properties on the proliferation of human RPTECs as a model for recovery from AKI. PEG diacrylate based hydrogels were generated with orthogonal control of mechanics and cell-substrate interactions. Using this platform, we found that increased substrate stiffness promotes RPTEC spreading and proliferation. RPTECs showed similar degrees of apoptosis and Yes-associated protein (YAP) nuclear localization regardless of stiffness, suggesting these were not key mediators of the effect. However, focal adhesion formation, cytoskeletal organization, focal adhesion kinase (FAK) activation, and extracellular signal-regulated kinase (ERK) activation were all enhanced with increasing substrate stiffness. Inhibition of ERK activation substantially attenuated the effect of stiffness on proliferation. In long-term culture, hydrogel stiffness promoted the formation of more complete epithelial monolayers with tight junctions, cell polarity, and an organized basement membrane. These data suggest that increased stiffness potentially may have beneficial consequences for the renal tubular epithelium during recovery from AKI.

Antagonistic Enzymes in a Biocatalytic pH Feedback System Program Autonomous DNA Hydrogel Life Cycles.

Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, nonlinear pH changes. The time scales are tunable by variation of the enzyme concentrations and additional buffer substances. The resulting material system operates in full autonomy after injection of the chemical fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.

Plant-Derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae.

Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects for which a tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harboring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver/reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC-EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF-DNA binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.