Red-Shifting B12-Dependent Photoreceptor Protein via Optical Coupling for Inducible Living Materials.

Cobalamin (B12)-dependent photoreceptors are gaining traction in materials synthetic biology, especially for optically controlling cell-to-cell adhesion in living materials. However, these proteins are mostly responsive to green light, limiting their deep-tissue applications. Here, we present a general strategy for shifting photoresponse of B12-dependent photoreceptor CarHC from green to red/far-red light via optical coupling. Using thiol-maleimide click chemistry, we labeled cysteine-containing CarHC mutants with SulfoCyanine5 (Cy5), a red light-capturing fluorophore. The resulting photoreceptors not only retained the ability to tetramerize in the presence of adenosylcobalamin (AdoB12), but also gained sensitivity to red light; labeled tetramers disassembled on red light exposure. Using genetically encoded click chemistry, we assembled the red-shifted proteins into hydrogels that degraded rapidly in response to red light. Furthermore, Saccharomyces cerevisiae cells were genetically engineered to display CarHC variants, which, alongside in situ Cy5 labeling, led to living materials that could assemble and disassemble in response to AdoB12 and red light, respectively. These results illustrate the CarHC spectrally tuned by optical coupling as a versatile motif for dynamically controlling cell-to-cell interactions within engineered living materials. Given their prevalence and ecological diversity in nature, this spectral tuning method will expand the use of B12-dependent photoreceptors in optogenetics and living materials.

B12-induced reassembly of split photoreceptor protein enables photoresponsive hydrogels with tunable mechanics.

Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.

Self-Assembly and Genetically Engineered Hydrogels.

The past few decades have witnessed the emergence of a variety of hydrogel materials for applications ranging from health to solutions for restoring natural environments. Among the numerous ways to synthesize these materials, directly assembling genetically engineered proteins into higher-order structures turned out to be a powerful strategy for hydrogel design. In recent years, the resulting genetically engineered (GE) hydrogels, noted for their modularity, versatility, and genetic programmability, are gaining traction with materials scientists and synthetic biologists who are eyeing the prospect of mass producing these materials via biosynthesis. In this chapter, we review the recent progresses in creating GE hydrogels, especially those enabled by self-assembling protein motifs, as well as their applications.

Injectable, photoresponsive hydrogels for delivering neuroprotective proteins enabled by metal-directed protein assembly.

Axon regeneration constitutes a fundamental challenge for regenerative neurobiology, which necessitates the use of tailor-made biomaterials for controllable delivery of cells and biomolecules. An increasingly popular approach for creating these materials is to directly assemble engineered proteins into high-order structures, a process that often relies on sophisticated protein chemistry. Here, we present a simple approach for creating injectable, photoresponsive hydrogels via metal-directed assembly of His6-tagged proteins. The B12-dependent photoreceptor protein CarHC can complex with transition metal ions through an amino-terminal His6-tag, which can further undergo a sol-gel transition upon addition of AdoB12, leading to the formation of hydrogels with marked injectability and photodegradability. The inducible phase transitions further enabled facile encapsulation and release of cells and proteins. Injecting the Zn2+-coordinated gels decorated with leukemia inhibitory factor into injured mouse optic nerves led to prolonged cellular signaling and enhanced axon regeneration. This study illustrates a powerful strategy for designing injectable biomaterials.

Cobalt-Cross-Linked, Redox-Responsive Spy Network Protein Hydrogels.

Although assembly of recombinant proteins by SpyTag/SpyCatcher chemistry has proven to be a versatile approach for creating bioactive hydrogels, the resulting Spy networks often exhibit weak mechanics due to the poor efficiency of interchain cross-linking. Here we leverage metal/ligand (i.e., cobalt/His6-tag) coordination interactions to modulate the bulk mechanics of the protein networks. The drastic difference between the Co2+ and Co3+ complexes in thermodynamic and kinetic properties enabled us to regulate the materials' properties and to immobilize and release recombinant proteins in a redox-dependent manner. The resulting hydrogels are capable of not only supporting cell growth and proliferation, but also influencing specific cell signaling via immobilized growth factors such as leukemia inhibitory factor (LIF). The integrated use of stimuli-responsive metal coordination and SpyTag/SpyCatcher chemistry opens up a new dimension for designing bioactive protein materials.

Synthesis of Entirely Protein-Based Hydrogels by Enzymatic Oxidation Enabling Water-Resistant Bioadhesion and Stem Cell Encapsulation.

Growing complexity in modern surgery and guided tissue repair calls for new materials capable of dual functions-water-resistant adhesion to biological tissues and stem cell encapsulation/delivery. Here, we demonstrate the creation of entirely recombinant protein-based adhesive hydrogels by leveraging the sequence derived from natural adhesive molecules-mussel foot protein-3 (Mfp-3), in vitro enzymatic oxidation mediated by recombinant tyrosinase and genetically encoded SpyTag/SpyCatcher chemistry. The resulting materials exhibited varied stiffness dependent on the polymer concentration, strong water-resistant adhesion to porcine skin, and excellent compatibility with 3D stem cell culture. The presence of SpyCatcher domains within the protein networks also enabled postgelation decoration with SpyTagged proteins under mild physiological conditions. These results point to a new approach for designing genetically programmable materials that enable both water-resistant bioadhesion and stem cell encapsulation for biomedical applications.

B12-Dependent Protein Oligomerization Facilitates Layer-by-Layer Growth of Photo/Thermal Responsive Nanofilms.

We report the robust growth of an entirely protein-based, photo- and thermoresponsive Layer-by-Layer nanofilm using genetically encoded SpyTag/SpyCatcher chemistry. The process was facilitated by AdoB12-induced tetramerization of photoreceptor proteins. Protein cargos can be released from the film in a light-dependent manner, showing its potential for therapeutic protein delivery.

Genetically Programming Stress-Relaxation Behavior in Entirely Protein-Based Molecular Networks.

We report the synthesis of a series of elastin-like polypeptide (ELP)-based molecular networks through the combined use of the covalent bond-forming SpyTag/SpyCatcher chemistry, physically entangled p53dim domains (Xs), and site-directed mutagenesis. The resulting networks shared similar chemical composition but differed significantly in their viscoelasticity. These materials exhibited excellent compatibility toward encapsulated fibroblasts and stem cells. These results point to a versatile strategy for designing viscoelastic materials by tapping into diverse protein-protein interactions.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Protein Hydrogel Microbeads for Selective Uranium Mining from Seawater.

Practical methods for oceanic uranium extraction have yet to be developed in order to tap into the vast uranium reserve in the ocean as an alternative energy. Here we present a protein hydrogel system containing a network of recently engineered super uranyl binding proteins (SUPs) that is assembled through thiol-maleimide click chemistry under mild conditions. Monodisperse SUP hydrogel microbeads fabricated by a microfluidic device further enable uranyl (UO22+) enrichment from natural seawater with great efficiency (enrichment index, K = 2.5 × 103) and selectivity. Our results demonstrate the feasibility of using protein hydrogels to extract uranium from the ocean.

Arteriovenous Fistula Creation by Nephrologist Access Surgeons Worldwide.

Several years ago, we published an article in this journal entitled "Autologous Arteriovenous Fistula Creation by Nephrologists." The goal of that effort was to review outcomes published by nephrologist access surgeons from around the world. An attempt was also made to define the elements that were necessary for successful autologous fistula creation in hopes that they might be used by others in an effort to increase the number of and durability of autologous fistulas in the dialysis population. Our goal, here, is to update the previous information based on recently published literature to better understand the role that nephrologist access surgeons play in the care of our dialysis patients worldwide.

Differential vulnerabilities of urethral afferents in diabetes and discovery of a novel urethra-to-urethra reflex.

Urethral reflexes are important regulators of micturition, and impairment of urethral afferent neuronal function may disrupt coordinated bladder and urethral activity, thereby contributing to voiding dysfunction in lower urinary tract disorders. Chemical stimulation by intraurethral irritant solution perfusion was used to determine whether urethral afferent neuronal function is altered in diabetes mellitus (DM). Sprague-Dawley rats were studied 10 wk after streptozotocin injection to induce DM or vehicle alone. Escalating doses of capsaicin (0.1-30 microM) or acetic acid (0.01-1%; AA) were perfused intraurethrally while recording isovolumetric bladder activity, urethral perfusion pressure, and electromyography of the external urethral sphincter (EUS-EMG). Some rats were additionally treated with alpha-bungarotoxin, hexamethonium, or bilateral transection of the sensory branches of the pudendal nerves (PudSNx). Intraurethral capsaicin inhibited bladder contractions in six out of seven control rats but not in any of six DM rats. Low-frequency oscillations (LFOs) of intraurethral pressure were observed in five out of six control rats with capsaicin-induced bladder inhibition. In contrast, intraurethral AA inhibited bladder contractions and enhanced tonic EUS-EMG activity in six out of six control and five out of six DM rats. LFOs occurred in four out of six control and three of five DM rats with AA-induced bladder inhibition. Chemically induced bladder inhibition and LFOs were not prevented by alpha-bungarotoxin but were eliminated by PudSNx and hexamethonium. Finally, LFOs were followed by phasic EUS activity. These findings show that DM affects urethral afferent neurons differentially, compromising those expressing TRPV1 receptors. Urethral smooth muscle LFOs are neurogenically mediated and induce EUS activity, revealing the existence of a hitherto undescribed reflex pathway: a smooth-to-striated muscle urethra-to-urethra reflex.

In vivo selection of tumor-targeting RNA motifs.

In an effort to target the in vivo context of tumor-specific moieties, we screened a large library of nuclease-resistant RNA oligonucleotides in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to p68, an RNA helicase that has been shown to be upregulated in colorectal cancer.

Voltage-dependent potassium currents of urethral afferent neurons in diabetes mellitus.

Urethra-to-bladder and urethra-to-urethra reflexes appear to be important for coordination of proper voiding. Diabetes mellitus (DM) is known to result in afferent neuropathy. Neuropathic alterations in electrophysiological properties of urethral afferent neurons may therefore contribute to voiding dysfunction seen in diabetes mellitus. Accordingly, we studied urethral afferent neuronal somata in streptozotocin-induced DM or age-matched vehicle controls by whole-cell patch clamp at 5- or 10-week time points. One week prior to study, Fast Blue was injected into the proximal urethra to label urethral afferent neurons. A previously undescribed diminution of afferent neuronal voltage-dependent potassium currents was a prominent feature of urethral afferent neuropathy in DM, acting to increase neuronal excitability. Thus, unlike bladder afferent neurons, urethral afferent neurons may be hyperexcitable well into DM progression.

Diabetic urethropathy compounds the effects of diabetic cystopathy.

PURPOSE: The effects of short-term and long-term diabetes mellitus on urethral function were investigated to determine the contribution of urethral dysfunction to diabetes mellitus voiding dysfunction. MATERIALS AND METHODS: Isovolumetric bladder pressure, urethral perfusion pressure and external urethral sphincter electromyography were measured in urethane anesthetized, female Sprague-Dawley rats (Charles River Laboratories, Wilmington, Massachusetts) 5 or 10 weeks after streptozotocin induced diabetes mellitus. Urethral responses to serial administration of the skeletal muscle blocker alpha-bungarotoxin, the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine and the alpha-adrenergic agonist L-phenylephrine were determined in diabetes mellitus and age matched controls. RESULTS: Peak bladder pressures and contraction amplitudes were significantly decreased in diabetes mellitus rats. Detrusor-sphincter dyssynergia occurred in approximately 30% of diabetes mellitus rats but never in controls. Alpha-Bungarotoxin caused a greater decrease in baseline urethral perfusion pressure in diabetes mellitus rats than in controls (approximately 40% vs approximately 15%). Bladder contraction associated urethral smooth muscle relaxation amplitudes were significantly less in diabetes mellitus rats than in controls. N(omega)-nitro-L-arginine significantly suppressed urethral relaxation in controls but not in diabetes mellitus rats. L-phenylephrine significantly increased baseline urethral perfusion pressure in diabetes mellitus rats but not in controls. The unassociated conditions of insensitivity to N-nitro-L-arginine and hypersensitivity to L-phenylephrine were more common in 10-week diabetes mellitus rats than in control rats. CONCLUSIONS: Diabetes mellitus induced urethropathy is characterized by external urethral sphincter dysfunction, decreased urethral smooth muscle relaxation and nitric oxide responsiveness, and increased urethral smooth muscle responsiveness to alpha(1)-adrenergic agonists. These changes increase outlet resistance and, thereby, decrease voiding efficiency. This exacerbates voiding dysfunction, creating a vicious cycle of progressive lower urinary tract damage and dysfunction. Early intervention targeting outlet resistance may be indicated.

Nitric oxide represses the Arabidopsis floral transition.

The correct timing of flowering is essential for plants to maximize reproductive success and is controlled by environmental and endogenous signals. We report that nitric oxide (NO) repressed the floral transition in Arabidopsis thaliana. Plants treated with NO, as well as a mutant overproducing NO (nox1), flowered late, whereas a mutant producing less NO (nos1) flowered early. NO suppressed CONSTANS and GIGANTEA gene expression and enhanced FLOWERING LOCUS C expression, which indicated that NO regulates the photoperiod and autonomous pathways. Because NO is induced by environmental stimuli and constitutively produced, it may integrate both external and internal cues into the floral decision.