The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

Nonspherical ultrasound microbubbles.

Surface tension provides microbubbles (MB) with a perfect spherical shape. Here, we demonstrate that MB can be engineered to be nonspherical, endowing them with unique features for biomedical applications. Anisotropic MB were generated via one-dimensionally stretching spherical poly(butyl cyanoacrylate) MB above their glass transition temperature. Compared to their spherical counterparts, nonspherical polymeric MB displayed superior performance in multiple ways, including i) increased margination behavior in blood vessel-like flow chambers, ii) reduced macrophage uptake in vitro, iii) prolonged circulation time in vivo, and iv) enhanced blood-brain barrier (BBB) permeation in vivo upon combination with transcranial focused ultrasound (FUS). Our studies identify shape as a design parameter in the MB landscape, and they provide a rational and robust framework for further exploring the application of anisotropic MB for ultrasound-enhanced drug delivery and imaging applications.

A native chemical chaperone in the human eye lens.

Cataract is one of the most prevalent protein aggregation disorders and still the most common cause of vision loss worldwide. The metabolically quiescent core region of the human lens lacks cellular or protein turnover; it has therefore evolved remarkable mechanisms to resist light-scattering protein aggregation for a lifetime. We now report that one such mechanism involves an unusually abundant lens metabolite, myo-inositol, suppressing aggregation of lens crystallins. We quantified aggregation suppression using our previously well-characterized in vitro aggregation assays of oxidation-mimicking human γD-crystallin variants and investigated myo-inositol's molecular mechanism of action using solution NMR, negative-stain TEM, differential scanning fluorometry, thermal scanning Raman spectroscopy, turbidimetry in redox buffers, and free thiol quantitation. Unlike many known chemical chaperones, myo-inositol's primary target was not the native, unfolded, or final aggregated states of the protein; rather, we propose that it was the rate-limiting bimolecular step on the aggregation pathway. Given recent metabolomic evidence that it is severely depleted in human cataractous lenses compared to age-matched controls, we suggest that maintaining or restoring healthy levels of myo-inositol in the lens may be a simple, safe, and globally accessible strategy to prevent or delay lens opacification due to age-onset cataract.

Raman spectroscopy reveals high phloem sugar content in leaves of canopy red oak trees.

A robust understanding of phloem functioning in tall trees evades us because current methods for collecting phloem sap do not lend themselves to measuring actively photosynthesizing canopy leaves. We show that Raman spectroscopy can be used as a quantitative tool to assess sucrose concentration in leaf samples. Specifically, we found that Raman spectroscopy can predict physiologically relevant sucrose concentrations (adjusted R2 of 0.9) in frozen leaf extract spiked with sucrose. We then apply this method to estimate sieve element sucrose concentration in rapidly frozen petioles of canopy red oak (Quercus rubra) trees and found that sucrose concentrations are > 1100 mM at midday and midnight. This concentration is predicted to generate a sieve element turgor pressure high enough to generate bulk flow through the phloem, but is potentially too high to allow for sucrose diffusion from photosynthetic cells. Our findings support the Münch hypothesis for phloem transport once the carbon is in the phloem and challenge the passive-loading hypothesis for carbon movement into the phloem for red oak. This study provides the first ˜in-situ (frozen in the functioning state) source sieve element sucrose concentration characterization in any plant, opening a new avenue for investigation of phloem functioning.

Establishment of perpendicular protrusion of type I collagen on TiO2 nanotube surface as a priming site of peri-implant connective fibers.

Natural teeth are supported by connective tissue collagen fibers that insert perpendicularly in the tooth cementum. Perpendicular insertion plays an important role in the maintenance of the junction between the oral epithelium and the periodontal connective tissue. Most titanium dental implant surfaces have no micro or macro structure to support perpendicularly oriented collagen attachment. Without this tight biologic seal to resist bacterial invasion and epithelial downgrowth, progressive bone loss in peri-implantitis is seen around dental implants. The purpose of this study was to establish the perpendicularly oriented collagen attachment to titanium oxide nanotube (TNT), and to assess its binding stability. TNT was prepared on the titanium-surface by anodization. Scanning electron microscopy (SEM) showed a regularly aligned TNT with an average 67 nm-diameter when anodized at 30 V for 3 h. Subsequently, collagen type I (CoI) was electrophoretically fused to anodic TNT in native polyacrylamide gel system where negatively charged CoI-C term was perpendicularly navigated to TNT. SEM and atomic force microscopy (AFM) were used to analyze CoI on the TiO2 and TNT surface. Several tens of nanometers of CoI protrusion were recorded by AFM. These protrusions may be long enough to be priming sites for cell-secreted CoI. CoI laid parallel to the titanium surface when fused by a chemical linker. Binding resistance of CoI against drastic ultrasonication was measured by Fourier-transform infrared spectroscopy attenuated total reflection (FTIR-ATR). The electrophoretically fused CoI in the titanium nanotube (TNT-CoIEPF) showed the significantly greatest binding resistance than the other groups (P < 0.01, a 1-way ANOVA and Tukey HSD post hoc test). Furthermore, TNT-CoIEPF surface rejected epithelial cell stretching and epithelial sheet formation. Chemically linked horizontal CoI on titanium oxide (TiO2) facilitated epithelial cell stretching and sheet formation.

The Coupled Straintronic-Photothermic Effect.

We describe the coupled straintronic-photothermic effect where coupling between bandgap of the 2D layered semiconductor under localized strains, optical absorption and the photo-thermal effect results in a large chromatic mechanical response in TMD-nanocomposites. Under the irradiation of visible light (405 nm to 808 nm), such locally strained atomic thin films based on 2H-MoS2 embedded in an elastomer such as poly (dimethyl) siloxane matrix exhibited a large amplitude of photo-thermal actuation compared to their unstrained counterparts. Moreover, the locally strain engineered nanocomposites showed tunable mechanical response giving rise to higher mechanical stress at lower photon energies. Scanning photoluminescence spectroscopy revealed a change in bandgap of 30 meV between regions encompassing highly strained compared to the unstrained few layers. For 1.6% change in the bandgap, the macroscopic photo-thermal response increased by a factor of two. Millimeter scale bending actuators based on the locally strained 2H-MoS2 resulted in significantly enhanced photo-thermal actuation displacements compared to their unstrained counterparts at lower photon energies and operated up to 30 Hz. Almost 1 mN photo-activated force was obtained at 50 mW and provided long-term stability. This study demonstrates a new mechanism in TMD-nanocomposites that would be useful for developing broad range of transducers.

Reactivity of a stable copper-dioxygen complex.

We report the isolation of a room temperature stable dipyrromethene Cu(O2) complex featuring a side-on O2 coordination. Reactivity studies highlight the unique ability of the dioxygen adduct for both hydrogen-atom abstraction and acid/base chemistry towards phenols, demonstrating that side-on superoxide species can be reactive entities.

Controlling the Roughness of Langmuir-Blodgett Monolayers.

Controlling the surface roughness of thin films with nanoscale precision is of significant interest for the rational design of surface coatings. Although wrinkling and buckling of Langmuir monolayers under compression has been demonstrated for several years, there is currently no method to precisely control this behavior during compression and thereby modify the surface roughness of deposited films. Here, we combine conventional Langmuir phase analysis with a novel dynamic viscoelasticity measurement to simply and accurately observe the jamming transition of monolayers of silica spheres, graphene oxide, and surfactant. By overcompressing beyond this point, the surface roughness of the deposited monolayer can be precisely controlled. This technique could be used to tune the surface properties of a variety of materials from lipids to nanoparticles.

Intracellular Delivery Using Nanosecond-Laser Excitation of Large-Area Plasmonic Substrates.

Efficiently delivering functional cargo to millions of cells on the time scale of minutes will revolutionize gene therapy, drug discovery, and high-throughput screening. Recent studies of intracellular delivery with thermoplasmonic structured surfaces show promising results but in most cases require time- or cost-intensive fabrication or lead to unreproducible surfaces. We designed and fabricated large-area (14 × 14 mm), photolithography-based, template-stripped plasmonic substrates that are nanosecond laser-activated to form transient pores in cells for cargo entry. We optimized fabrication to produce plasmonic structures that are ultrasmooth and precisely patterned over large areas. We used flow cytometry to characterize the delivery efficiency of cargos ranging in size from 0.6 to 2000 kDa to cells (up to 95% for the smallest molecule) and viability of cells (up to 98%). This technique offers a throughput of 50000 cells/min, which can be scaled up as necessary. This technique is also cost-effective as each large-area photolithography substrate can be used to deliver cargo to millions of cells, and switching to a nanosecond laser makes the setup cheaper and easier to use. The approach we present offers additional desirable features: spatial selectivity, reproducibility, minimal residual fragments, and cost-effective fabrication. This research supports the development of safer genetic and viral disease therapies as well as research tools for fundamental biological research that rely on effectively delivering molecules to millions of living cells.

Facet-Selective Epitaxy of Compound Semiconductors on Faceted Silicon Nanowires.

Integration of compound semiconductors with silicon (Si) has been a long-standing goal for the semiconductor industry, as direct band gap compound semiconductors offer, for example, attractive photonic properties not possible with Si devices. However, mismatches in lattice constant, thermal expansion coefficient, and polarity between Si and compound semiconductors render growth of epitaxial heterostructures challenging. Nanowires (NWs) are a promising platform for the integration of Si and compound semiconductors since their limited surface area can alleviate such material mismatch issues. Here, we demonstrate facet-selective growth of cadmium sulfide (CdS) on Si NWs. Aberration-corrected transmission electron microscopy analysis shows that crystalline CdS is grown epitaxially on the {111} and {110} surface facets of the Si NWs but that the Si{113} facets remain bare. Further analysis of CdS on Si NWs grown at higher deposition rates to yield a conformal shell reveals a thin oxide layer on the Si{113} facet. This observation and control experiments suggest that facet-selective growth is enabled by the formation of an oxide, which prevents subsequent shell growth on the Si{113} NW facets. Further studies of facet-selective epitaxial growth of CdS shells on micro-to-mesoscale wires, which allows tuning of the lateral width of the compound semiconductor layer without lithographic patterning, and InP shell growth on Si NWs demonstrate the generality of our growth technique. In addition, photoluminescence imaging and spectroscopy show that the epitaxial shells display strong and clean band edge emission, confirming their high photonic quality, and thus suggesting that facet-selective epitaxy on NW substrates represents a promising route to integration of compound semiconductors on Si.

Molecular orientation of asphaltenes and PAH model compounds in Langmuir-Blodgett films using sum frequency generation spectroscopy.

Asphaltenes are an important class of compounds in crude oil whose surface activity is important for establishing reservoir rock wettability which impacts reservoir drainage. While many phenomenological interfacial studies with crude oils and asphaltenes have been reported, there is very little known about the molecular level interactions between asphaltenes and mineral surfaces. In this study, we analyze Langmuir-Blodgett films of asphaltenes and related model compounds with sum frequency generation (SFG) vibrational spectroscopy. In SFG, the polarization of the input (vis, IR) and output (SFG) beams can be varied, which allows the orientation of different functional groups at the interface to be determined. SFG clearly indicates that asphaltene polycyclic aromatic hydrocarbons (PAHs) are highly oriented in the plane of the interface and that the peripheral alkanes are transverse to the interface. In contrast, model compounds with oxygen functionality have PAHs oriented transverse to the interface. Computational quantum chemistry is used to support corresponding band assignments, enabling robust determination of functional group orientations.

Peering at a buried polymer-crystal interface: probing heterogeneous nucleation by sum frequency generation vibrational spectroscopy.

Sum frequency generation vibrational spectroscopy (SFG-VS) has been applied to investigate the selective crystallization of two forms of acetaminophen (ACM) on polymer surfaces. To our knowledge, this is the first account of SFG-VS being applied to study a polymer-crystal interface. SFG elucidates the molecular-level interactions governing phase selection at this buried interface, providing insight into the process of polymer-induced heteronucleation (PIHn) in solution as well as from the vapor phase. ACM heteronucleates from supersaturated aqueous solution in the metastable orthorhombic crystal form on poly(methyl methacrylate) (PMMA) surfaces, whereas the thermodynamically stable monoclinic crystal form is observed to form on poly(n-butyl methacrylate) (PBMA) surfaces. When the ACM crystals were grown by sublimation, only the monoclinic form was observed on both PMMA and PBMA. SFG-VS results indicate that hydrogen bonds are formed between PMMA C═O groups and the orthorhombic ACM crystals at the PMMA-ACM interface. At PBMA-monoclinic ACM interfaces, no hydrogen bond formation was observed. This research demonstrates that SFG-VS can be used to probe molecular interactions at polymer-crystal interfaces. Understanding the interfacial molecular interactions will ultimately provide a rational basis for improving methods for polymorph discovery and selection based on heteronucleation on polymer surfaces.

Deducing 2D crystal structure at the liquid/solid interface with atomic resolution: a combined STM and SFG study.

Sum frequency generation vibrational spectroscopy (SFG) has been applied to study two-dimensional (2D) crystals formed by an isophthalic acid diester on the surface of highly oriented pyrolytic graphite, providing complementary measurements to scanning tunneling microscopy (STM) and computational modeling. SFG results indicate that both aromatic and C=O groups in the 2D crystal tilt from the surface. This study demonstrates that a combination of SFG and STM techniques can be used to gain a more complete picture of 2D crystal structure, and it is necessary to consider solvent-2D crystal interactions and dynamics in the computer models to achieve an accurate representation of interfacial structure.

Detection of tethered biocide moiety segregation to silicone surface using sum frequency generation vibrational spectroscopy.

Polymer surface properties are controlled by the molecular surface structures. Sum frequency generation (SFG) vibrational spectroscopy has been demonstrated to be a powerful technique to study polymer surface structures at the molecular level in different chemical environments. In this research, SFG has been used to study the surface segregation of biocide moieties derived from triclosan (TCS) and tetradecyldimethyl (3-trimethoxysilylpropyl) ammonium chloride (C-14 QAS) that have been covalently bound to a poly(dimethylsiloxane) (PDMS) matrix. PDMS materials are being developed as coatings to control biofouling. This SFG study indicated that TCS-moieties segregate to the surface when the bulk concentration of TCS-moieties exceeds 8.75% by weight. Surface segregation of C-14 QAS moieties was detected after 5% by weight incorporation into a PDMS matrix. SFG results were found to correlate well with antifouling activity, providing a molecular interpretation of such results. This research showed that SFG can aid in the development of coatings for controlling biofouling by elucidating the chemical structure of the coating surface.

Solventless adhesive bonding using reactive polymer coatings.

A novel solventless adhesive bonding (SAB) process is reported, which is applicable to a wide range of materials including, but not limited to, poly(dimethylsiloxane) (PDMS). The bonding is achieved through reactions between two complementary polymer coatings, poly(4-aminomethyl-p-xylylene-co-p-xylylene) and poly(4-formyl-p-xylylene-co-p-xylylene), which are prepared by chemical vapor deposition (CVD) polymerization of the corresponding [2.2]paracyclophanes and can be deposited on complementary microfluidic units to be bonded. These CVD-based polymer films form well-adherent coatings on a range of different substrate materials including polymers, glass, silicon, metals, or paper and can be stored for extended periods prior to bonding without losing their bonding capability. Tensile stress data are measured on PDMS with various substrates and compared favorably to current methods such as oxygen plasma and UV/ozone. Sum frequency generation (SFG) has been used to probe the presence of amine and aldehyde groups on the surface after CVD polymerization and their conversion during bonding. In addition to bonding, unreacted functional groups present on the luminal surface of microfluidic channels provide free chemical groups for further surface modification. Fluorescently labeled molecules including rhodamine-conjugated streptavidin and atto-655 NHS ester were used to verify the presence of active functional groups on the luminal surfaces after bonding.

Ultrafast hot-carrier dynamics at chemically modified Ge interfaces probed by SHG.

Time-resolved second-harmonic generation (SHG) was used to study the hot-carrier dynamics and nonlinear optical properties of S-terminated and Cl-terminated Ge(111) interfaces on the femtosecond time scale. The hot-carrier second-order nonlinear optical susceptibilities were determined to be 720 +/- 50 times greater than the valence-band second-order nonlinear optical susceptibilities for the Ge(111)-S system and 880 +/- 100 times greater in the Ge(111)-Cl system. Furthermore, the ground- and excited-state second-order nonlinear optical susceptibilities are suggested to be out of phase for Ge(111)-S and Ge(111)-Cl systems, leading to a pump-induced decrease in the SHG signal as opposed to the increase in the SHG signal observed in the Ge(111)-GeO2 system. Although the SHG response reaches a steady state in 415 +/- 90 fs in the Ge(111)-GeO2 system, a faster response is observed in the Ge(111)-S system, 220 +/- 85 fs, and in the Ge(111)-Cl system, 172 +/- 50 fs. This suggests significantly faster carrier cooling at the Ge(111)-Cl and Ge(111)-S interfaces, with significant implications for hot-carrier mediated device degradation, and migration to high-K dielectrics.