Transpupillary-Guided Trans-Scleral Transplantation of Subretinal Grafts in a Retinal Degeneration Mouse Model.

Transplantation of photoreceptor cells and retinal pigment epithelial (RPE) cells provide a potential therapy for retinal degeneration diseases. Subretinal transplantation of therapeutic donor cells into mouse recipients is challenging due to the limited surgical space allowed by the small volume of the mouse eye. We developed a trans-scleral surgical transplantation platform with direct transpupillary vision guidance to facilitate the subretinal delivery of exogenous cells in mouse recipients. The platform was tested using retinal cell suspensions and three-dimensional retinal sheets collected from rod-rich Rho::EGFP mice and cone-rich OPN1LW-EGFP;NRL-/- mice, respectively. Live/dead assay showed low cell mortality for both forms of donor cells. Retinal grafts were successfully delivered into the subretinal space of a mouse model of retinal degeneration, Rd1/NS, with minimum surgical complications as detected by multimodal confocal scanning laser ophthalmoscope (cSLO) imaging. Two months post-transplantation, histological staining demonstrated evidence of advanced maturation of the retinal grafts into 'adult' rods and cones (by robust Rho::EGFP, S-opsin, and OPN1LW:EGFP expression, respectively) in the subretinal space. Here, we provide a surgical platform that can enable highly accurate subretinal delivery with a low rate of complications in mouse recipients. This technique offers precision and relative ease of skill acquisition. Furthermore, the technique could be used not only for studies of subretinal cell transplantation but also for other intraocular therapeutic studies including gene therapies.

Factors Affecting Stability of RNA - Temperature, Length, Concentration, pH, and Buffering Species.

RNA is prone to both chemical degradation and/or physical instability. Some of the factors affecting stability of RNA in solution are its length, 3' poly A tail and 5' cap integrity, excipients, buffering species, pH of the solution, nucleases, and divalent cations. In this work, we showed the effect of temperature, messenger RNA (mRNA) length, buffering species, pH of the solution, and the concentration of mRNA on its chemical and physical stability. Our thermodynamic analysis of a 4000 nucleotide-long mRNA measured an activation energy of 31.5 kcal/mol normalized per phosphodiester backbone. We found mRNA length to be negatively correlated to its stability. Buffering species and pH of the solution affected mRNA integrity along with affecting the onset temperature of melting obtained by Differential Scanning Calorimetry (DSC) thermograms. It was also found that increasing the concentration of mRNA in solution increased its stability.

Antiresorptive Medications Prior to Stereotactic Body Radiotherapy for Spinal Metastasis are Associated with Reduced Incidence of Vertebral Body Compression Fracture.

STUDY DESIGN: Retrospective Cohort. OBJECTIVE: Antiresorptive drugs are often given to minimize fracture risk for bone metastases, but data regarding optimal time or ability to reduce stereotactic body radiotherapy (SBRT)-induced fracture risk is limited. This study examines the association between antiresorptive use surrounding spinal SBRT and vertebral compression fracture (VCF) incidence to provide information regarding effectiveness and optimal timing of use. METHODS: Patients treated with SBRT for spinal metastases at a single institution between 2009-2020 were included. Kaplan-Meier analysis was used to compare cumulative incidence of VCF for those taking antiresorptive drugs pre-SBRT, post-SBRT only, and none at all. Cox proportional hazards and Fine-Gray competing risk models were used to identify additional factors associated with VCF. RESULTS: Of the 234 patients (410 vertebrae) analyzed, 49 (20.9%) were taking bisphosphonates alone, 42 (17.9%) were taking denosumab alone, and 25 (10.7%) were taking both. Kaplan-Meier analysis revealed a statistically significant lower VCF incidence for patients initiating antiresorptive drugs before SBRT compared to those taking none at all (4% vs 12% at 1 year post-SBRT, P = .045; and 4% vs 23% at 2 years, P = .008). On multivariate analysis, denosumab duration (HR: .87, P = .378) or dose (HR: 1.00, P = .644) as well as bisphosphonate duration (HR: .98, P= .739) or dose (HR: .99, P= .741) did not have statistical significance on VCF incidence. CONCLUSION: Initiating antiresorptive agents before SBRT may reduce the risk of treatment-induced VCF. Antiresorptive drugs are underutilized in patients with spine metastases and may represent a useful intervention to minimize toxicity and improve long-term outcomes.

CaMKII binds both substrates and activators at the active site.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a signaling protein required for long-term memory. When activated by Ca2+/CaM, it sustains activity even after the Ca2+ dissipates. In addition to the well-known autophosphorylation-mediated mechanism, interaction with specific binding partners also persistently activates CaMKII. A long-standing model invokes two distinct S and T sites. If an interactor binds at the T-site, then it will preclude autoinhibition and allow substrates to be phosphorylated at the S site. Here, we specifically test this model with X-ray crystallography, molecular dynamics simulations, and biochemistry. Our data are inconsistent with this model. Co-crystal structures of four different activators or substrates show that they all bind to a single continuous site across the kinase domain. We propose a mechanistic model where persistent CaMKII activity is facilitated by high-affinity binding partners that kinetically compete with autoinhibition by the regulatory segment to allow substrate phosphorylation.

From Protein Design to the Energy Landscape of a Cold Unfolding Protein.

Understanding protein folding is crucial for protein sciences. The conformational spaces and energy landscapes of cold (unfolded) protein states, as well as the associated transitions, are hardly explored. Furthermore, it is not known how structure relates to the cooperativity of cold transitions, if cold and heat unfolded states are thermodynamically similar, and if cold states play important roles for protein function. We created the cold unfolding 4-helix bundle DCUB1 with a de novo designed bipartite hydrophilic/hydrophobic core featuring a hydrogen bond network which extends across the bundle in order to study the relative importance of hydrophobic versus hydrophilic protein-water interactions for cold unfolding. Structural and thermodynamic characterization resulted in the discovery of a complex energy landscape for cold transitions, while the heat unfolded state is a random coil. Below ∼0 °C, the core of DCUB1 disintegrates in a largely cooperative manner, while a near-native helical content is retained. The resulting cold core-unfolded state is compact and features extensive internal dynamics. Below -5 °C, two additional cold transitions are seen, that is, (i) the formation of a water-mediated, compact, and highly dynamic dimer, and (ii) the onset of cold helix unfolding decoupled from cold core unfolding. Our results suggest that cold unfolding is initiated by the intrusion of water into the hydrophilic core network and that cooperativity can be tuned by varying the number of core hydrogen bond networks. Protein design has proven to be invaluable to explore the energy landscapes of cold states and to robustly test related theories.

2D Gadolinium Oxide Nanoplates as T1 Magnetic Resonance Imaging Contrast Agents.

Millions of people a year receive magnetic resonance imaging (MRI) contrast agents for the diagnosis of conditions as diverse as fatty liver disease and cancer. Gadolinium chelates, which provide preferred T1 contrast, are the current standard but face an uncertain future due to increasing concerns about their nephrogenic toxicity as well as poor performance in high-field MRI scanners. Gadolinium-containing nanocrystals are interesting alternatives as they bypass the kidneys and can offer the possibility of both intracellular accumulation and active targeting. Nanocrystal contrast performance is notably limited, however, as their organic coatings block water from close interactions with surface Gadoliniums. Here, these steric barriers to water exchange are minimized through shape engineering of plate-like nanocrystals that possess accessible Gadoliniums at their edges. Sulfonated surface polymers promote second-sphere relaxation processes that contribute remarkable contrast even at the highest fields (r1 = 32.6 × 10-3 m Gd-1 s-1 at 9.4 T). These noncytotoxic materials release no detectable free Gadolinium even under mild acidic conditions. They preferentially accumulate in the liver of mice with a circulation half-life 50% longer than commercial agents. These features allow these T1 MRI contrast agents to be applied for the first time to the ex vivo detection of nonalcoholic fatty liver disease in mice.

Electronically integrated, mass-manufactured, microscopic robots.

Fifty years of Moore's law scaling in microelectronics have brought remarkable opportunities for the rapidly evolving field of microscopic robotics1-5. Electronic, magnetic and optical systems now offer an unprecedented combination of complexity, small size and low cost6,7, and could be readily appropriated for robots that are smaller than the resolution limit of human vision (less than a hundred micrometres)8-11. However, a major roadblock exists: there is no micrometre-scale actuator system that seamlessly integrates with semiconductor processing and responds to standard electronic control signals. Here we overcome this barrier by developing a new class of voltage-controllable electrochemical actuators that operate at low voltages (200 microvolts), low power (10 nanowatts) and are completely compatible with silicon processing. To demonstrate their potential, we develop lithographic fabrication-and-release protocols to prototype sub-hundred-micrometre walking robots. Every step in this process is performed in parallel, allowing us to produce over one million robots per four-inch wafer. These results are an important advance towards mass-manufactured, silicon-based, functional robots that are too small to be resolved by the naked eye.

Characterization of CaMKIIα holoenzyme stability.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent Tm = 36°C), which is slightly stabilized by ATP/MgCl2 binding (apparent Tm = 40°C) and significantly stabilized by regulatory segment binding (apparent Tm = 60°C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent Tm ~ 90°C), and the holoenzyme structure has multiple unfolding transitions ranging from ~60°C to 100°C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact.

Magnetic handshake materials as a scale-invariant platform for programmed self-assembly.

Programmable self-assembly of smart, digital, and structurally complex materials from simple components at size scales from the macro to the nano remains a long-standing goal of material science. Here, we introduce a platform based on magnetic encoding of information to drive programmable self-assembly that works across length scales. Our building blocks consist of panels with different patterns of magnetic dipoles that are capable of specific binding. Because the ratios of the different panel-binding energies are scale-invariant, this approach can, in principle, be applied down to the nanometer scale. Using a centimeter-sized version of these panels, we demonstrate 3 canonical hallmarks of assembly: controlled polymerization of individual building blocks; assembly of 1-dimensional strands made of panels connected by elastic backbones into secondary structures; and hierarchical assembly of 2-dimensional nets into 3-dimensional objects. We envision that magnetic encoding of assembly instructions into primary structures of panels, strands, and nets will lead to the formation of secondary and even tertiary structures that transmit information, act as mechanical elements, or function as machines on scales ranging from the nano to the macro.

Atomic Layer Deposition for Membranes, Metamaterials, and Mechanisms.

Bending and folding techniques such as origami and kirigami enable the scale-invariant design of 3D structures, metamaterials, and robots from 2D starting materials. These design principles are especially valuable for small systems because most micro- and nanofabrication involves lithographic patterning of planar materials. Ultrathin films of inorganic materials serve as an ideal substrate for the fabrication of flexible microsystems because they possess high intrinsic strength, are not susceptible to plasticity, and are easily integrated into microfabrication processes. Here, atomic layer deposition (ALD) is employed to synthesize films down to 2 nm thickness to create membranes, metamaterials, and machines with micrometer-scale dimensions. Two materials are studied as model systems: ultrathin SiO2 and Pt. In this thickness limit, ALD films of these materials behave elastically and can be fabricated with fJ-scale bending stiffnesses. Further, ALD membranes are utilized to design micrometer-scale mechanical metamaterials and magnetically actuated 3D devices. These results establish thin ALD films as a scalable basis for micrometer-scale actuators and robotics.

Audio cues enhance mirroring of arm motion when visual cues are scarce.

Swing in a crew boat, a good jazz riff, a fluid conversation: these tasks require extracting sensory information about how others flow in order to mimic and respond. To determine what factors influence coordination, we build an environment to manipulate incoming sensory information by combining virtual reality and motion capture. We study how people mirror the motion of a human avatar's arm as we occlude the avatar. We efficiently map the transition from successful mirroring to failure using Gaussian process regression. Then, we determine the change in behaviour when we introduce audio cues with a frequency proportional to the speed of the avatar's hand or train individuals with a practice session. Remarkably, audio cues extend the range of successful mirroring to regimes where visual information is sparse. Such cues could facilitate joint coordination when navigating visually occluded environments, improve reaction speed in human-computer interfaces or measure altered physiological states and disease.

Kirigami Mechanics as Stress Relief by Elastic Charges.

We develop a geometric approach to understand the mechanics of perforated thin elastic sheets, using the method of strain-dependent image elastic charges. This technique recognizes the buckling response of a hole under an external load as a geometrically tuned mechanism of stress relief. We use a diagonally pulled square paper frame as a model system to quantitatively test and validate our approach. Specifically, we compare nonlinear force-extension curves and global displacement fields in theory and experiment. We find a strong softening of the force response accompanied by curvature localization at the inner corners of the buckled frame. Counterintuitively, though in complete agreement with our theory, for a range of intermediate hole sizes, wider frames are found to buckle more easily than narrower ones. Upon extending these ideas to many holes, we demonstrate that interacting elastic image charges can provide a useful kirigami design principle to selectively relax stresses in elastic materials.

Nonlinear mechanics of thin frames.

The dramatic effect kirigami, such as hole cutting, has on the elastic properties of thin sheets invites a study of the mechanics of thin elastic frames under an external load. Such frames can be thought of as modular elements needed to build any kirigami pattern. Here we develop the technique of elastic charges to address a variety of elastic problems involving thin sheets with perforations, focusing on frames with sharp corners. We find that holes generate elastic defects (partial disclinations), which act as sources of geometric incompatibility. Numerical and analytic studies are made of three different aspects of loaded frames-the deformed configuration itself, the effective mechanical properties in the form of force-extension curves, and the buckling transition triggered by defects. This allows us to understand generic kirigami mechanics in terms of a set of force-dependent elastic charges with long-range interactions.

Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants.

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the ß1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

Study of the PDK1/AKT signaling pathway using selective PDK1 inhibitors, HCS, and enhanced biochemical assays.

The PI3K/AKT signaling pathway has an important regulatory role in cancer cell growth and tumorigenesis. Signal transduction through this pathway requires the assembly and activation of PDK1 and AKT at the plasma membrane. On activation of the pathway, PDK1 and AKT1/2 translocate to the membrane and bind to phosphatidylinositol-(3,4,5)-trisphosphate (PIP(3)) through interaction with their pleckstrin-homology domains. A biochemical method was developed to measure the kinase activity of PDK1 and AKT1/2, utilizing nickel-chelating coated lipid vesicles as a way to mimic the membrane environment. The presence of these vesicles in the reaction buffer enhanced the specific activity of the His-tagged PDK1 (full-length, and the truncated kinase domain) and the activity of the full-length His-tagged AKT1 and AKT2 when assayed in a cascade-type reaction. This enhanced biochemical assay is also suitable for measuring the inhibition of PDK1 by several selective compounds from the carbonyl-4-amino-pyrrolopyrimidine (CAP) series. One of these inhibitors, PF-5168899, was further evaluated using a high content cell-based assay in the presence of CHO cells engineered with GFP-PDK1.

Challenges and approaches for assay development of membrane and membrane-associated proteins in drug discovery.

In addition to its role as a barrier between the cytoplasm and the extracellular milieu, the cell membrane is a scaffold for a diverse collection of receptors and enzymes. The organization afforded by this scaffold serves to ensure an efficient interaction between the components of the membrane. The desire to maintain this organization in solution is a challenge for the appropriate interrogation of these biochemical components. This chapter will discuss strategies that allow biochemical analysis of membrane-associated enzymes within standard biochemical reactions. The advantages of these screening strategies in identifying valuable compounds from compound libraries and in understanding the intricacies of complex multiprotein complexes (i.e., chemotaxis) will be discussed.

Template-directed self-assembly enhances RTK catalytic domain function.

Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface. Autophosphorylation of template-assembled tyrosine kinase domains from the insulin, EphB2, and Tie2 receptors led to substantially larger phosphorylation levels compared with domains assayed under conventional conditions. Template-directed assembly increased the total substrate phosphorylation of the insulin and EphB2 receptor kinase domains as much as 60-fold and 15-fold, respectively. In contrast, substrate phosphorylation by template-assembled Tie2 was much lower than conventional conditions. The lower activity observed with the template is more biologically relevant because autophosphorylation of Tie2 is self-inhibitory. These results, as well as the underlying similarity between the organization of template-assembled and natural membrane signaling environments, suggest that template-directed assembly of signaling proteins will provide widespread benefit to basic and applied signal transduction research, especially drug discovery.

Template-directed assembly of signaling proteins: a novel drug screening and research tool.

A multitude of proteins reside at or near the cell membrane, which provides a unique environment for organizing and promoting assemblies of proteins that are involved in a variety of cellular signaling functions. Many of these proteins and pathways are implicated in disease. For example, strong links have been established between receptor tyrosine kinases and disease, most notably, cancer. However, a significant impediment to researchers remains: membrane-associated proteins are difficult to reconstitute and study. Template-directed assembly represents a powerful new technology that enables the assembly of membrane-associated proteins. We show that template-directed assembly restores tyrosine kinase activity and regulation, and provides a way for researchers to build multicomponent assemblies. As an example of better enzyme regulation, the Tie2 tyrosine kinase domain exhibits (biologically relevant) autoinhibitory behavior when template assembled. Also, template-assembled insulin receptor tyrosine kinase domains exhibit significant autophosphorylation (none detected without template-directed assembly) and an eightfold increase in substrate phosphorylation (compared to best solution conditions). Thus, template-directed assembly has a demonstrated ability to effectively produce more biologically relevant results using these commercial reagents. Template-directed assembly promises to be generally applicable to the signaling networks important for human health, because these pathways frequently contain membrane-associated proteins that require the organizing influence of a membrane surface.