Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.

Spatial Atlas for Mapping Vascular Microcalcification Using 18F-NaF PET/CT: Application in Hyperphosphatemic Familial Tumoral Calcinosis.

BACKGROUND: Vascular calcification causes significant morbidity and occurs frequently in diseases of calcium/phosphate imbalance. Radiolabeled sodium fluoride positron emission tomography/computed tomography has emerged as a sensitive and specific method for detecting and quantifying active microcalcifications. We developed a novel technique to quantify and map total vasculature microcalcification to a common space, allowing simultaneous assessment of global disease burden and precise tracking of site-specific microcalcifications across time and individuals. METHODS: To develop this technique, 4 patients with hyperphosphatemic familial tumoral calcinosis, a monogenic disorder of FGF23 (fibroblast growth factor-23) deficiency with a high prevalence of vascular calcification, underwent radiolabeled sodium fluoride positron emission tomography/computed tomography imaging. One patient received serial imaging 1 year after treatment with an IL-1 (interleukin-1) antagonist. A radiolabeled sodium fluoride-based microcalcification score, as well as calcification volume, was computed at all perpendicular slices, which were then mapped onto a standardized vascular atlas. Segment-wise mCSmean and mCSmax were computed to compare microcalcification score levels at predefined vascular segments within subjects. RESULTS: Patients with hyperphosphatemic familial tumoral calcinosis had notable peaks in microcalcification score near the aortic bifurcation and distal femoral arteries, compared with a control subject who had uniform distribution of vascular radiolabeled sodium fluoride uptake. This technique also identified microcalcification in a 17-year-old patient, who had no computed tomography-defined calcification. This technique could not only detect a decrease in microcalcification score throughout the patient treated with an IL-1 antagonist but it also identified anatomic areas that had increased responsiveness while there was no change in computed tomography-defined macrocalcification after treatment. CONCLUSIONS: This technique affords the ability to visualize spatial patterns of the active microcalcification process in the peripheral vasculature. Further, this technique affords the ability to track microcalcifications at precise locations not only across time but also across subjects. This technique is readily adaptable to other diseases of vascular calcification and may represent a significant advance in the field of vascular biology.

Direct Laser Writing of Silica Nanoparticle Nanocomposites: Probing Mechanical Reinforcement and Understanding Structural Color from Design Parameters.

Nanocomposite materials have been thoroughly exploited in additive manufacturing, as a means to alter physical, chemical, and optical properties of resulting structures. Herein, nanocomposite materials suitable for direct laser writing (DLW) by two-photon polymerization are presented. These materials, comprising silica nanoparticles, bring significant added value to the technology through physical reinforcement and controllable photonic properties. Incorporation into acrylate photoresists, via a one-step fabrication process, enables the formation of complex structures with large overhangs. The inclusion of 150 nm silica nanoparticles in DLW photoresists at high concentrations, allows for the fabrication of composite microstructures that show reflected color, a product of the relative contributions from the quasi-ordering and random scattering. Using common DLW design parameters, such as slicing distance and structure dimension, a wide gamut of structural color, in solution, using a set concentration of nanoparticles is demonstrated. Numerical modeling is employed to predict the reflected wavelength of the pixel arrays, across the visible spectrum, and this information is used to encode reflected colors into different pixel arrays.

Room Temperature Fabrication of Macroporous Lignin Membranes for the Scalable Production of Black Silicon.

Rising global demand for biodegradable materials and green sources of energy has brought attention to lignin. Herein, we report a method for manufacturing standalone lignin membranes without additives for the first time to date. We demonstrate a scalable method for macroporous (∼100 to 200 nm pores) lignin membrane production using four different organosolv lignin materials under a humid environment (>50% relative humidity) at ambient temperatures (∼20 °C). A range of different thicknesses is reported with densely porous films observed to form if the membrane thickness is below 100 nm. The fabricated membranes were readily used as a template for Ni2+ incorporation to produce a nickel oxide membrane after UV/ozone treatment. The resultant mask was etched via an inductively coupled plasma reactive ion etch process, forming a silicon membrane and as a result yielding black silicon (BSi) with a pore depth of >1 µm after 3 min with reflectance <3% in the visible light region. We anticipate that our lignin membrane methodology can be readily applied to various processes ranging from catalysis to sensing and adapted to large-scale manufacturing.

Expression of N-Terminal Cysteine Aß42 and Conjugation to Generate Fluorescent and Biotinylated Aß42.

Fluorescent derivatives of the ß-amyloid peptides (Aß) are valuable tools for studying the interactions of Aß with cells. Facile access to labeled expressed Aß offers the promise of Aß with greater sequence and stereochemical integrity, without impurities from amino acid deletion and epimerization. Here, we report methods for the expression of Aß42 with an N-terminal cysteine residue, Aß(C1-42), and its conjugation to generate Aß42 bearing fluorophores or biotin. The methods rely on the hitherto unrecognized observation that expression of the Aß(MC1-42) gene yields the Aß(C1-42) peptide, because the N-terminal methionine is endogenously excised by Escherichia coli. Conjugation of Aß(C1-42) with maleimide-functionalized fluorophores or biotin affords the N-terminally labeled Aß42. The expression affords ∼14 mg of N-terminal cysteine Aß from 1 L of bacterial culture. Subsequent conjugation affords ∼3 mg of labeled Aß from 1 L of bacterial culture with minimal cost for labeling reagents. High-performance liquid chromatography analysis indicates the N-terminal cysteine Aß to be >97% pure and labeled Aß peptides to be 94-97% pure. Biophysical studies show that the labeled Aß peptides behave like unlabeled Aß and suggest that labeling of the N-terminus does not substantially alter the properties of the Aß. We further demonstrate applications of the fluorophore-labeled Aß peptides by using fluorescence microscopy to visualize their interactions with mammalian cells and bacteria. We anticipate that these methods will provide researchers convenient access to useful N-terminally labeled Aß, as well as Aß with an N-terminal cysteine that enables further functionalization.

Large-Area Fabrication of Vertical Silicon Nanotube Arrays via Toroidal Micelle Self-Assembly.

We present a highly scalable, room-temperature strategy for fabricating vertical silicon nanotube arrays derived from a toroidal micelle pattern via a water vapor-induced block copolymer (BCP) self-assembly mechanism. A polystyrene-b-poly(ethylene oxide) (PS-b-PEO) BCP system can be self-assembled into toroidal micelle structures (diameter: 400-600 nm) on a PS-OH-modified substrate in a facile manner contrasting with other complex processes described in the literature. It was found that a minimum PS-b-PEO thickness of ∼86 nm is required for the toroidal self-assembly. Furthermore, a water vapor annealing treatment at room conditions (∼25 °C, 60 min) is shown to vastly enhance the ordering of micellar structures. A liquid-phase infiltration process was used to generate arrays of iron and nickel oxide nanorings. These oxide structures were used as templates for pattern transfer into the underlying silicon substrate via plasma etching, resulting in large-area 3D silicon nanotube arrays. The overall simplicity of this technique, as well as the wide potential versatility of the resulting metal structures, proves that such room-temperature synthesis routes are a viable pathway for complex nanostructure fabrication, with potential applicability in fields such as optics or catalysis.

Assessment of atherosclerosis in multiple myeloma and smoldering myeloma patients using 18F- sodium fluoride PET/CT.

BACKGROUND: To compare the NaF uptake in the thoracic aorta and whole heart, as an early indicator of atherosclerosis, in multiple myeloma (MM) and smoldering multiple myeloma (SMM) patients with a healthy control (HC) group. METHODS: Forty-four untreated myeloma patients (35 MM and nine SMM) and twenty-six age and gender-matched HC subjects were collected. Each individual's NaF uptake in three parts of the aorta (AA: ascending aorta, AR: aortic arch, DA: descending aorta) and the whole heart was segmented. Average global standardized uptake value means were derived by sum of the product of each slice area divided by the sum of those slice areas. Results were reported as target to background ratio (TBR). RESULTS: There was a significant difference between the NaF uptake in the thoracic aorta of myeloma and HC groups [AA (myeloma = 1.82 ± 0.21, HC = 1.24 ± 0.02), AR (myeloma = 1.71 ± 0.19, HC = 1.28 ± 0.03) and DA (myeloma = 1.96 ± 0.28, HC = 1.38 ± 0.03); P-values < 0.001]. The difference in the whole heart NaF uptake between two groups was also significant (P < 0.001). CONCLUSIONS: We observed a higher uptake of NaF in the thoracic aorta and whole heart of myeloma patients in comparison to the matched control group.

MECP2 duplication syndrome in a patient from Cameroon.

MECP2 duplication syndrome (MDS; OMIM 300260) is an X-linked neurodevelopmental disorder caused by nonrecurrent duplications of the Xq28 region involving the gene methyl-CpG-binding protein 2 (MECP2; OMIM 300005). The core phenotype of affected individuals includes infantile hypotonia, severe intellectual disability, very poor-to-absent speech, progressive spasticity, seizures, and recurrent infections. The condition is 100% penetrant in males, with observed variability in phenotypic expression within and between families. Features of MDS in individuals of African descent are not well known. Here, we describe a male patient from Cameroon, with MDS caused by an inherited 610 kb microduplication of Xq28 encompassing the genes MECP2, IRAK1, L1CAM, and SLC6A8. This report supplements the public data on MDS and contributes by highlighting the phenotype of this condition in affected individuals of African descent.

Precise Definition of a "Monolayer Point" in Polymer Brush Films for Fabricating Highly Coherent TiO2 Thin Films by Vapor-Phase Infiltration.

In this work, we show that in order to fabricate coherent titania (TiO2) films with precise thickness control, it is critical to generate a complete polymer brush monolayer. To date, demonstrations of such dense polymer monolayer formation that can be utilized for inorganic infiltration have been elusive. We describe a versatile bottom-up approach to covalently and rapidly (60 s processing) graft hydroxyl-terminated poly(2-vinyl pyridine) (P2VP-OH) polymers on silicon substrates. P2VP-OH monolayer films of varying thicknesses can subsequently be used to fabricate high-quality TiO2 films. Our innovative strategy is based upon room-temperature titanium vapor-phase infiltration of the grafted P2VP-OH polymer brushes that can produce TiO2 nanofilms of 2-4 nm thicknesses. Crucial parameters are explored, including molecular weight and solution concentration for grafting dense P2VP-OH monolayers from the liquid phase with high coverage and uniformity across wafer-scale areas (>2 cm2). Additionally, we compare the P2VP-OH polymer systems with another reactive polymer, poly(methyl methacrylate)-OH, and a relatively nonreactive polymer, poly(styrene)-OH. Furthermore, we prove the latter to be effective for surface blocking and deactivation. We show a simple process to graft monolayers for polymers that are weakly interacting with one another but more challenging for reactive systems. Our methodology provides new insight into the rapid grafting of polymer brushes and their ability to form TiO2 films. We believe that the results described herein are important for further expanding the use of reactive and unreactive polymers for fields including area-selective deposition, solar cell absorber layers, and antimicrobial surface coatings.

Electrochemical Sensing of Hydrogen Peroxide Using Block Copolymer Templated Iron Oxide Nanopatterns.

A new enzyme-free sensor based on iron oxide (Fe3O4) nanodots fabricated on an indium tin oxide (ITO) substrate via a block copolymer template was developed for highly sensitive and selective detection of hydrogen peroxide (H2O2). The self-assembly-based process described here for Fe3O4 formation is a simple, cost-effective, and reproducible process. The H2O2 response of the fabricated electrodes was linear from 2.5 × 10-3 to 6.5 mM with a sensitivity of 191.6 µA mM-1cm-2 and a detection limit of 1.1 × 10-3 mM. The electrocatalytic activity of Fe3O4 nanodots toward the electroreduction of H2O2 was described by cyclic voltammetric and amperometric techniques. The sensor described here has a strong anti-interference ability to a variety of common biological and inorganic substances.

Large Block Copolymer Self-Assembly for Fabrication of Subwavelength Nanostructures for Applications in Optics.

Nanostructured surfaces are common in nature and exhibit properties such as antireflectivity (moth eyes), self-cleaning (lotus leaf), iridescent colors (butterfly wings), and water harvesting (desert beetles). We now understand such properties and can mimic some of these natural structures in the laboratory. However, these synthetic structures are limited since they are not easily mass produced over large areas due to the limited scalability of current technologies such as UV-lithography, the high cost of infrastructure, and the difficulty in nonplanar surfaces. Here, we report a solution process based on block copolymer (BCP) self-assembly to fabricate subwavelength structures on large areas of optical and curved surfaces with feature sizes and spacings designed to efficiently scatter visible light. Si nanopillars (SiNPs) with diameters of ∼115 ± 19 nm, periodicity of 180 ± 18 nm, and aspect ratio of 2-15 show a reduction in reflectivity by a factor of 100, <0.16% between 400 and 900 nm at an angle of incidence of 30°. Significantly, the reflectivity remains below 1.75% up to incident angles of 75°. Modeling the efficiency of a SiNP PV suggests a 24.6% increase in efficiency, representing a 3.52% (absolute) or 16.7% (relative) increase in electrical energy output from the PV system compared to AR-coated device.

Nanoscale silicon substrate patterns from self-assembly of cylinder forming poly(styrene)-block-poly(dimethylsiloxane) block copolymer on silane functionalized surfaces.

Poly(styrene)-block-poly(dimethylsiloxane) (PS-b-PDMS) is an excellent block copolymer (BCP) system for self-assembly and inorganic template fabrication because of its high Flory-Huggins parameter (χ âˆ¼ 0.26) at room temperature in comparison to other BCPs, and high selective etch contrast between PS and PDMS block for nanopatterning. In this work, self-assembly in PS-b-PDMS BCP is achieved by combining hydroxyl-terminated poly(dimethylsiloxane) (PDMS-OH) brush surfaces with solvent vapor annealing. As an alternative to standard brush chemistry, we report a simple method based on the use of surfaces functionalized with silane-based self-assembled monolayers (SAMs). A solution-based approach to SAM formation was adopted in this investigation. The influence of the SAM-modified surfaces upon BCP films was compared with polymer brush-based surfaces. The cylinder forming PS-b-PDMS BCP and PDMS-OH polymer brush were synthesized by sequential living anionic polymerization. It was observed that silane SAMs provided the appropriate surface chemistry which, when combined with solvent annealing, led to microphase segregation in the BCP. It was also demonstrated that orientation of the PDMS cylinders may be controlled by judicious choice of the appropriate silane. The PDMS patterns were successfully used as an on-chip etch mask to transfer the BCP pattern to underlying silicon substrate with sub-25 nm silicon nanoscale features. This alternative SAM/BCP approach to nanopattern formation shows promising results, pertinent in the field of nanotechnology, and with much potential for application, such as in the fabrication of nanoimprint lithography stamps, nanofluidic devices or in narrow and multilevel interconnected lines.

Area Selective Polymer Brush Deposition.

Polymer brush films with chemical functionality to attach to site specific substrate areas are introduced for area selective deposition (ASD) application. It is demonstrated that polymer brushes with chemically defined end sites can be selectively bound to copper-specific regions of patterned copper/silica (Cu/SiO2 ) substrates. The process described overcomes various limitations of currently used technology including cost, complexity, and throughput, with potential implications for future electronic devices and nanomanufacturing. A comparative study of amine-terminated polystyrene and amine-terminated poly-2-vinyl pyridine polymer brushes (i.e., PS-NH2 and P2VP-NH2 ) with similar molecular weights display contrasting behavior on patterned Cu/SiO2 line features. Further, a thiol terminated poly-2-vinyl pyridine polymer brush (i.e., P2VP-SH) is investigated as a direct spin-on process to fabricate a metal oxide layer atop Cu areas only. The results presented here detail a novel methodology and open a new exciting process for ASD practices that can facilitate the precise deposition of dense metal, semiconductor, or dielectric films. We also discuss the applicability of polymer brushes to ASD uses going forward.

Controlled solvent vapor annealing of a high χ block copolymer thin film.

Molecular self-assembling block copolymers (BCPs) have shown promise as a next generation bottom-up lithography technology. However, a critical step in advancing this approach is the elimination of polymer dewetting due to bulk solvent nucleation and thermodynamically driven film rupture that can occur during the solvent vapor annealing process. We report on the pattern formation via phase segregation of spin coated diblock copolymer films through the investigation of annealing parameters in the limit of high solvent vapor saturation conditions that results in wafer-scale patterning without observing polymer dewetting defects. Specifically, the work addresses polymer dewetting in diblock copolymer nanodot templates through the use of a "neutral" functionalization layer and the development of a custom-built solvent vapor annealing chamber to precisely control saturation conditions. Furthermore, the long anneal times (4 h) using a standard static solvent vapor annealing procedure were reduced to ∼15-30 minutes with our dynamic solvent vapor annealing system for the high χ, cylindrical forming poly(styrene)-block-poly(4-vinyl-pyridine) [PS-b-P4VP] diblock copolymer system. We discuss the kinetic mechanism governing the phase segregation process that highlights the small processing window bounded by long phase segregation timescales (≳1 min) on one side and the initiation of polymer film dewetting on the other. These results demonstrate a key step towards realizing a high fidelity, low cost BCP patterning technique for large-scale "bottom-up" feature definition at nanometer length scales.

Morphological evolution of lamellar forming polystyrene-block-poly(4-vinylpyridine) copolymers under solvent annealing.

In this work, we are reporting a very simple and efficient method to form lamellar structures of symmetric polystyrene-block-poly(4-vinylpyridine) (PS-b-P4VP) copolymer thin films with vertically (to the surface plane) orientated lamellae using a solvent annealing approach. The methodology does not require any brush chemistry to engineer a neutral surface and it is the block neutral nature of the film-solvent vapour interface that defines the orientation of the lamellae. The microphase separated structure of two different molecular weight lamellar forming PS-block-P4VP copolymers formed under solvent vapour annealing was monitored using atomic force microscopy (AFM) so as to understand the morphological changes of the films upon different solvent exposure. In particular, the morphology changes from micellar structures to well-defined microphase separated arrangements. The choice of solvent/s (single and dual solvent exposure) and the solvent annealing conditions (temperature, time etc.) has important effects on structural transitions of the films and it was found that a block neutral solvent was required to realize vertically aligned P4VP lamellae. The results of the structural variation of the phase separated nanostructured films through the exposure to ethanol are also described.

In-depth TEM characterization of block copolymer pattern transfer at germanium surfaces.

Dry plasma etching for the pattern transfer of mask features is fundamental to semiconductor processing and the development of device and electrically conducting elements becomes more challenging as features reach the deep nanoscale regime. In this work, high resolution transmission electron microscopy (TEM) coupled with energy dispersive x-ray (EDX) characterization were used to analyze the pattern transfer of graphoepitaxially aligned block copolymer (BCP) features to germanium (Ge) substrates as a function of time. The BCP patterns were converted into metal oxide hardmasks in order to affect good aspect ratios of the transferred features. An unusual interface layer between metal oxide nanowires and the germanium-on-insulator substrate was observed. EDX analysis shows that the origin of this interface layer is a result of the presence of a negative tone e-beam resist material, HSQ (hydrogen silsesquioxane). HSQ was employed as a guiding material to align line-space features of poly(styrene)-block-poly(4-vinylpyridine) (PS-b-P4VP) BCP with 16 nm half-pitch topography. Additionally, the existence of a metal oxide layer (from the initial PS-b-P4VP film) is also shown through ex situ TEM and EDX characterization. Three dimensional modeling of features is also provided giving a unique insight into the arrangement and structure of BCP features prior to and after the pattern transfer process. The results presented in this article highlight the accuracy of high resolution electron microscopy and elemental mapping of BCP generated on-chip etch masks to observe and understand through-film features affecting pattern transfer.

In-situ observations of nanoscale effects in germanium nanowire growth with ternary eutectic alloys.

Vapour-liquid-solid (VLS) techniques are popular routes for the scalable synthesis of semiconductor nanowires. In this article, in-situ electron microscopy is used to correlate the equilibrium content of ternary (Au0.75 Ag0.25 -Ge and Au0.65 Ag0.35 -Ge) metastable alloys with the kinetics, thermodynamics and diameter of Ge nanowires grown via a VLS mechanism. The shape and geometry of the heterogeneous interfaces between the liquid eutectic and solid Ge nanowires varies as a function of nanowire diameter and eutectic alloy composition. The behaviour of the faceted heterogeneous liquid-solid interface correlates with the growth kinetics of the nanowires, where the main growth facet at the solid nanowire-liquid catalyst drop contact line lengthens for faster nanowire growth kinetics. Pronounced diameter dependent growth kinetics, as inferred from liquid-solid interfacial behaviour, is apparent for the synthesised nanowires. Direct in-situ microscopy observations facilitates the comparison between the nanowire growth behaviour from ternary (Au-Ag-Ge) and binary (Au-Ge) eutectic systems.

Solvent vapor annealing of block copolymers in confined topographies: commensurability considerations for nanolithography.

The directed self-assembly of block copolymer (BCP) materials in topographically patterned substrates (i.e., graphoepitaxy) is a potential methodology for the continued scaling of nanoelectronic device technologies. In this Communication, an unusual feature size variation in BCP nanodomains under confinement with graphoepitaxially aligned cylinder-forming poly(styrene)-block-poly(4-vinylpyridine) (PS-b-P4VP) BCP is reported. Graphoepitaxy of PS-b-P4VP BCP line patterns (CII ) is accomplished via topo-graphy in hydrogen silsequioxane (HSQ) modified substrates and solvent vapor annealing (SVA). Interestingly, reduced domain sizes in features close to the HSQ guiding features are observed. The feature size reduction is evident after inclusion of alumina into the P4VP domains followed by pattern transfer to the silicon substrate. It is suggested that this nano-domain size perturbation is due to solvent swelling effects during SVA. It is proposed that using a commensurability value close to the solvent vapor annealed periodicity will alleviate this issue leading to uniform nanofins.

Nanoscale neuroelectrode modification via sub-20 nm silicon nanowires through self-assembly of block copolymers.

Neuroprosthetic technologies for therapeutic neuromodulation have seen major advances in recent years but these advances have been impeded due to electrode failure or a temporal deterioration in the device recording or electrical stimulation potential. This deterioration is attributed to an intrinsic host tissue response, namely glial scarring or gliosis, which prevents the injured neurons from sprouting, drives neurite processes away from the neuroelectrode and increases signal impedance by increasing the distance between the electrode and its target neurons. To address this problem, there is a clinical need to reduce tissue encapsulation of the electrodes in situ and improve long-term neuroelectrode function. Nanotopographical modification has emerged as a potent methodology for the disruption of protein adsorption and cellular adhesion in vitro. This study investigates the use of block copolymer self-assembly technique for the generation of sub-20 nm nanowire features on silicon substrates. Critically, these nanostructures were observed to significantly reduce electrical impedance and increase conductivity. Human neuroblastoma SH-SY5Y cells cultured on nanowire substrates for up to 14 days were associated with enhanced focal adhesion reinforcement and a reduction in proliferation. We conclude that nanowire surface modulation may offer significant potential as an electrode functionalization strategy.