Reversible and irreversible inhibitors of coronavirus Nsp15 endoribonuclease.

The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.

Towards better understanding the stiffness of nanocomposites via parametric study of an analytical model modeling parameters and experiments.

The stiffness of polymeric materials can be improved dramatically with the addition of nanoparticles. In theory, as the nanoparticle loading in the polymer increases, the nanocomposite becomes stiffer; however, experiments suggest that little or no stiffness improvement is observed beyond an optimal nanoparticle loading. The mismatch between the theoretical and experimental findings, particularly at high particle loadings, needs to be understood for the effective use of nanoparticles. In this respect, we have recently developed an analytical model to close the gap in the literature and predict elastic modulus of nanocomposites. The model is based on a three-phase Mori-Tanaka model coupled with the Monte-Carlo method, and satisfactorily captures the experimental results, even at high nanoparticle loadings. The developed model can also be used to study the effects of agglomeration in nanocomposites. In this paper, we use this model to study the effects of agglomeration and related model parameters on the stiffness of nanocomposites. In particular, the effects of particle orientation, critical distance, dispersion state and agglomerate property, and particle aspect ratio are investigated to demonstrate capabilities of the model and to observe how optimal particle loading changes with respect these parameters. The study shows that the critical distance defining agglomerates and the properties of agglomerates are the key design parameters at high particle loadings. These two parameters rule the optimal elastic modulus with respect to particle loading. The findings will allow researchers to form design curves and successfully predict the elastic moduli of nanocomposites without the exhaustive experimental undertakings.

Nanoparticle-based surface enhanced Raman spectroscopic imaging of biological arrays.

Surfaces serve as the communication link between the adsorbate and the substrate. Hence, a thorough understanding of the surface chemistries directly interfacing with biological molecules and other adsorbates would provide insight into the fabrication approach as well as the adsorption characteristics of biomolecules adsorbed on the surface. This paper presents a surface-enhanced Raman spectroscopy (SERS) method for high-sensitivity detection and reading of protein microarrays based on gold nanoparticle labels. The reagent employed was 30 nm gold nanoparticles modified with a bifunctional Raman reporter molecule, 5,5'-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB), to integrate anti-bovine IgG for an antigen response in the immunoassay and generate an intense SERS signal. The signal from the DSNB reporter molecule, particularly the strong symmetric nitro stretch was used for the detection of antigen-antibody interactions. Issues related to the sensitivity and selectivity of the assay were also addressed. This work provides useful insights into SERS-based immunoassays and serves as the basis for an eventful adventure into interfacial biomolecular interactions.

A predictive model towards understanding the effect of reinforcement agglomeration on the stiffness of nanocomposites.

Nanocomposite technologies can be significantly enhanced through a careful exploration of the effects of agglomerates on mechanical properties. Existing models are either overly simplified (e.g., neglect agglomeration effects) or often require a significant amount of computational resources. In this study, a novel continuum-based model with a statistical approach was developed. The model is based on a modified three-phase Mori-Tanaka model, which accounts for the filler, agglomerate, and matrix regions. Fillers are randomly dispersed in a defined space to predict agglomeration tendency. The proposed model demonstrates good agreement with the experimentally measured elastic moduli of spin-coated cellulose nanocrystal reinforced polyamide-6 films. The techniques and methodologies presented in the study are sufficiently general in that they can be extended to the analyses of various types of polymeric nanocomposite systems.

Application of Surface-Enhanced Raman Spectroscopy to Guide Therapy for Advanced Prostate Cancer Patients.

A critical unmet need for advanced prostate cancer (PCa) patients is optimizing systemic treatments to maximize the benefit for individuals. The response of patients with metastatic castration-resistant prostate cancer (mCRPC) to androgen receptor (AR)-directed hormonal treatments (i.e., enzalutamide and abiraterone) is mediated by the expression of a molecular variant of the androgen receptor called androgen receptor variant 7 (AR-V7). Detection and measurement of AR-V7 in mCRPC patients will lead to more informed PCa treatment. Herein, we demonstrate a quantitative nanoparticle-enhanced sandwich antibody assay for the successful ex vivo measurement of AR-V7 protein in serum from mCRPC patients. The nanoparticles are constructed as extrinsic Raman spectroscopy labels (ERLs), and surface-enhanced Raman spectroscopy (SERS) is used for assay readout. Our approach does not require specialized specimen collection materials, circulating tumor cell enrichment, or pretreatment of serum. Calibration of our assay is accomplished by expressing AR-V7 in an appropriate cell line as AR-V7 is not commercially available. We demonstrate a linear calibration curve from cell lysate and correlate lysate protein with mRNA from cultured prostate cancer cells. Finally, we demonstrate a novel pilot-scale application for clinical use by quantitatively measuring AR-V7 in serum of seven advanced PCa patients. Distinct separation of PCa patients by AR-V7 status (positive or negative) was observed. Together, the presence and amount of AR-V7 in serum offer predictive and prognostic value to inform selection between two classes of systemic treatments (i.e., hormones or taxanes). Triaging patients that are AR-V7-positive to other systemic treatments (e.g., taxane-based chemotherapy) can improve progression-free survival and overall survival.

Evaluation of the electroanalytical performance of carbon-on-gold films prepared by electron-beam evaporation.

Carbon film electrodes can often be used without pretreatment, and their fabrication allows for flexibility in size and shape and for mass production. In this work, we are exploring layered structures comprised of thin films of carbon on gold (eC/Au) prepared by electron-beam evaporation. These extremely flat films are not pyrolyzed and are comprised of mainly amorphous carbon but still exhibit reasonable conductivity due to the underlying gold layer. eC/Au electrodes, without any pretreatment, yield similar heterogeneous electron-transfer rates for benchmark redox systems and significantly lower background current in comparison with polished glassy carbon. Interestingly, they show insignificant adsorption of quinones, which is uncommon for most carbon electrode materials. However, eC/Au is still prone to adsorption of airborne hydrocarbons when exposed to ambient air like most graphitic materials. With reproducibly fast electron transfer kinetics, low background current, negligible adsorption, and ultraflat surface, eC/Au films are a promising candidate for electrochemical and sensor applications.

Gold nanostars as a colloidal substrate for in-solution SERS measurements using a handheld Raman spectrometer.

The evolution of Raman spectroscopy into a useful analytical technique has been due, in part, to the development of inexpensive, compact instrumentation and advancements in methodologies that enhance Raman intensities. Surface enhanced Raman scattering (SERS) is a primary methodology for quantitative and low detection limit measurements. While a broad array of applications using solid SERS substrates have been demonstrated, in-solution SERS measurements are not as widely pursued. This work seeks to optimize the synthesis of gold nanostars (AuNS) as a colloidal SERS substrate for in-solution measurements using handheld instrumentation. The types and concentrations of two buffers typically used for AuNS synthesis are examined to optimize the SERS intensity of a chemisorbed Raman probe. The observed SERS intensity primarily depends on conditions that allow higher surface coverage of the probe. Conditions that result in AuNS aggregates are found to be most optimal for SERS, similar to other nanoparticle shapes. A method to quantitate methimazole, an anti-hormone pharmaceutical, in urine is developed and reported. The primary impact of this work is the demonstration of the combination of water dispersible substrates and handheld instrumentation for rapid and sensitive analytical measurements.

Stretchable, tough, self-recoverable, and cytocompatible chitosan/cellulose nanocrystals/polyacrylamide hybrid hydrogels.

As medical practitioners' interest in hydrogels continues to grow, their new expectations in terms of mechanical properties, biocompatibility and durability are changed. Here, we demonstrated a new strategy to improve both mechanical properties and self-recovery of double network (DN) hydrogels by introducing a self-healing network, consisting of carboxymethyl chitosan (CMC) and dialdehyde cellulose nanocrystals (DACNC). Notably, the hydrogel could be repeatedly stretched to 4 times its initial length and has tensile strength of 244 kPa, and completely recovered its shape when compressed by 90% and had the compressive strength up to 8 MPa. In addition, the deformed hydrogel recovered 81.3% of its dissipated energy at room temperature without any external stimuli. The hydrogel also exhibited good biocompatibility. We have developed a new method to fabricate stretchable and tough hydrogels that could spontaneously self-repair following mechanical deformation. They are promising for controlled drug release and dye adsorption.

Immuno-impedimetric Biosensor for Onsite Monitoring of Ascospores and Forecasting of Sclerotinia Stem Rot of Canola.

Sclerotinia stem rot, caused by the fungal pathogen Sclerotinia sclerotiorum, is a destructive disease of canola and many other broadleaf crops. The primary inoculum responsible for initiating Sclerotinia epidemics is airborne ascospores released from the apothecia of sclerotia. Timely detection of the presence of airborne ascospores can serve as an early-warning system for forecasting and management of the disease. A major challenge is to develop a portable and automated device which can be deployed onsite to detect and quantify the presence of minute quantities of ascospores in the air and serves as a unit in a network of systems for forecasting of the epidemic. In this communication, we present the development of an impedimetric non-Faradaic biosensor based on anti-S. sclerotiorum polyclonal antibodies as probes to selectively capture the ascospores and sense their binding by an impedance based interdigitated electrode which was found to directly and unambiguously correlate the number of ascospores on sensor surface with the impedance response.

A surface plasmon resonance based inhibition immunoassay for measurement of steroid hormones.

Quantitative measurement of small-molecule metabolites is now emerging as an effective way to link the metabolite profile to disease state. Surface plasmon resonance (SPR) is a sensing platform that has demonstrated applicability for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. Herein, we utilized an indirect detection format and developed an inhibition immunoassay for the quantitative measurement of 17ß-estradiol (E2) using SPR. One competitor, BSA-E2 conjugate, was immobilized to the SPR chip via the reaction between the primary amino group of the conjugate and the succinimide group (NHS) introduced by the formation of a thiol-NHS monolayer on gold surface. Free E2 molecules compete with BSA-E2 on chip surface for binding sites provided by a monoclonal anti-E2 antibody. It was found the binding affinity of the antibody to BSA-E2 conjugate increases with decreasing surface coverage of BSA-E2 conjugate. Under optimal conditions, a sigmoidal calibration curve with a negative slope and a dynamic range from 10 pM to 2 nM was generated. The detection limit of the immunoassay is estimated to be 0.3 pM. Moreover, the immunoassay exhibits high specificity for E2 detection using estrone (E1) as a potential interference.

Functionalized gold nanoparticle-enhanced competitive assay for sensitive small-molecule metabolite detection using surface plasmon resonance.

The development of sensing systems for the measurement of small molecules is an active area of research. A sensor based approach for the measurement of metabolites can potentially provide the simplicity and portability required for widespread use. Rapid detection and quantitation of small-molecule metabolites can potentially emerge as an effective way to link the metabolite profile to the disease state. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. The work described here combines a periplasmic binding protein for recognition with target modified gold nanoparticles (AuNPs) in a competitive assay format for folic acid (FA) detection. Specifically, a SPR imaging substrate containing immobilized folate binding protein (FBP) is used to measure the adsorption of FA conjugated AuNPs. The immobilization of the FBP and the binding of the FA conjugated AuNPs are characterized and optimized. It is shown that free FA in solution can be quantitatively measured by competition for the surface binding sites with the functionalized AuNPs. We demonstrate that the dynamic range can be lowered from micromolar to nanomolar by simply decreasing the concentration of FA conjugated AuNPs, thus lowering the limit of detection to 2.9 nM. This type of competitive assay can be applied to a range of small molecules, which paves the way for future multiplexed analysis of metabolites using SPR.

Cellulose nanocrystal-derived hollow mesoporous carbon spheres and their application as a metal-free catalyst.

In this contribution, we demonstrate the fabrication of hollow mesoporous carbon spheres (HCSs) derived from cellulose nanocrystals (CNCs). The HCSs were prepared by templating CNCs onto sacrificial silica spheres followed by heat treatment. Mesoporous carbon spheres result from the removal of the silica spheres by etching. The walls of the HCSs are approximately 4 nm thick and are composed of amorphous and graphitic carbon. The catalytic activity of the HCSs was investigated for the reduction of 4-nitrophenol (4-NP) to 4-aminophenol (4-AP) by sodium borohydride (NaBH4). The present investigation reveals the outstanding catalytic activity of these particles. The reaction rate followed pseudo-first order kinetics with k value of 4.72 × 10-3 s-1 and activity parameter of 52.2 s-1 g-1, which showed superior performance compared to that of metal nanoparticle and metal nanoparticle-carbon hybrid based catalysts.

Humidity affects the morphology of particles emitted from beclomethasone dipropionate pressurized metered dose inhalers.

The effects of propellant type, cosolvent content, and air humidity on the morphology and solid phase of the particles produced from solution pressurized metered dose inhalers containing the corticosteroid beclomethasone dipropionate were investigated. The active ingredient was dissolved in the HFA propellants 134a and 227ea with varying levels of the cosolvent ethanol and filled into pressurized metered dose inhalers. Inhalers were actuated into an evaporation chamber under controlled temperature and humidity conditions and sampled using a single nozzle, single stage inertial impactor. Particle morphology was assessed qualitatively using field emission scanning electron microscopy and focused ion beam-helium ion microscopy. Drug solid phase was assessed using Raman microscopy. The relative humidity of the air during inhaler actuation was found to have a strong effect on the particle morphology, with solid spheroidal particles produced in dry air and highly porous particles produced at higher humidity levels. Air humidification was found to have no effect on the solid phase of the drug particles, which was predominantly amorphous for all tested formulations. A critical level of air relative humidity was required to generate porous particles for each tested formulation. This critical relative humidity was found to depend on the amount of ethanol used in the inhaler, but not on the type of propellant utilized. The results indicate that under the right circumstances water vapor saturation followed by nucleated water condensation or ice deposition occurs during particle formation from evaporating propellant-cosolvent-BDP droplets. This finding reveals the importance of condensed water or ice as a templating agent for porosity when particle formation occurs at saturated conditions, with possible implications on the pharmacokinetics of solution pMDIs and potential applications in particle engineering for drug delivery.

Fungal Isolate Optimized for Biogenesis of Silver Nanoparticles with Enhanced Colloidal Stability.

Understanding the synthesis and properties of nanomaterials is critical for reliable applications. Biological systems, such as fungi, have been described as a "green" alternative to synthesis, yet knowledge gaps exist in terms of production variability, comparison with commercial products, and identifying a clear biological advantage over other synthesis methods. In this study, we evaluated 12 fungal isolates of Fusarium oxysporum for Ag nanoparticle production and characterized the resultant biologically produced (biogenic) nanoparticles. We show evidence that isolate selection, temperature, and pH can influence the quantity, size, and shape of nanoparticles. All F. oxysporum isolates produced Ag nanoparticles, but in varied quantities. Increasing incubation temperature increased the quantity, yet nanoparticle diameter was inversely related to temperature. Variations in pH predominately influenced nanoparticle morphology. A direct comparison with commercial, chemically produced Ag nanoparticles yielded physical similarities; however, important differences in surface chemistry are observed. Biogenic nanoparticles show a greater degree of colloidal stability in high-ionic-strength solutions, pointing to a biological advantage associated with the fungal produced layer (corona) surrounding the nanoparticles. It is clear that understanding the organic layer and interfacial interactions will be beneficial in developing innovative applications, particularly in the field of biosensing.

Aryl Diazonium Chemistry for the Surface Functionalization of Glassy Biosensors.

Nanostring resonator and fiber-optics-based biosensors are of interest as they offer high sensitivity, real-time measurements and the ability to integrate with electronics. However, these devices are somewhat impaired by issues related to surface modification. Both nanostring resonators and photonic sensors employ glassy materials, which are incompatible with electrochemistry. A surface chemistry approach providing strong and stable adhesion to glassy surfaces is thus required. In this work, a diazonium salt induced aryl film grafting process is employed to modify a novel SiCN glassy material. Sandwich rabbit IgG binding assays are performed on the diazonium treated SiCN surfaces. Fluorescently labelled anti-rabbit IgG and anti-rabbit IgG conjugated gold nanoparticles were used as markers to demonstrate the absorption of anti-rabbit IgG and therefore verify the successful grafting of the aryl film. The results of the experiments support the effectiveness of diazonium chemistry for the surface functionalization of SiCN surfaces. This method is applicable to other types of glassy materials and potentially can be expanded to various nanomechanical and optical biosensors.

Diazonium Chemistry for the Bio-Functionalization of Glassy Nanostring Resonator Arrays.

Resonant glassy nanostrings have been employed for the detection of biomolecules. These devices offer high sensitivity and amenability to large array integration and multiplexed assays. Such a concept has however been impaired by the lack of stable and biocompatible linker chemistries. Diazonium salt reduction-induced aryl grafting is an aqueous-based process providing strong chemical adhesion. In this work, diazonium-based linker chemistry was performed for the first time on glassy nanostrings, which enabled the bio-functionalization of such devices. Large arrays of nanostrings with ultra-narrow widths down to 10 nm were fabricated employing electron beam lithography. Diazonium modification was first developed on SiCN surfaces and validated by X-ray photoelectron spectroscopy. Similarly modified nanostrings were then covalently functionalized with anti-rabbit IgG as a molecular probe. Specific enumeration of rabbit IgG was successfully performed through observation of downshifts of resonant frequencies. The specificity of this enumeration was confirmed through proper negative control experiments. Helium ion microscopy further verified the successful functionalization of nanostrings.

Specific detection of Campylobacter jejuni using the bacteriophage NCTC 12673 receptor binding protein as a probe.

Campylobacter jejuni is found in the intestines of poultry, cattle, swine, wild birds and pet animals and is the major cause of foodborne gastroenteritis in developed countries. We report the use of the receptor binding protein (RBP) of Campylobacter bacteriophage NCTC 12673 for the specific capture of Campylobacter jejuni bacteria using RBP-derivatized capturing surfaces. The Gp48 RBP was expressed as a glutathione S-transferase-Gp48 (GST-Gp48) fusion protein and immobilized onto surface plasmon resonance (SPR) surfaces using glutathione self-assembled monolayers (GSH SAM). Bovine serum albumin (BSA) was used to block any non-specific binding. Glutathione SAM leads to an oriented attachment of the protein, resulting in a two- to three-fold improvement of bacterial capture when compared to dithiobis(succinimidyl propionate) (DTSP) SAM-based unoriented attachment. The specificity of recognition was confirmed using Salmonella enterica subsp. enterica serovar Typhimurium as a negative control, which indeed showed negligible binding. The detection limit of the RBP-derivatized SPR surfaces was found to be 10(2) cfu/ml. Finally, GST-Gp48 was also immobilized onto magnetic beads that were successfully used to capture and pre-concentrate the host pathogen from suspension.

Diazonium-derived aryl films on gold nanoparticles: evidence for a carbon-gold covalent bond.

Tailoring the surface chemistry of metallic nanoparticles is generally a key step for their use in a wide range of applications. There are few examples of organic films covalently bound to metal nanoparticles. We demonstrate here that aryl films are formed on gold nanoparticles from the spontaneous reduction of diazonium salts. The structure and the bonding of the film is probed with surface-enhanced Raman scattering (SERS). Extinction spectroscopy and SERS show that a nitrobenzene film forms on gold nanoparticles from the corresponding diazonium salt. Comparison of the SERS spectrum with spectra computed from density functional theory models reveals a band characteristic of a Au-C stretch. The observation of this stretch is direct evidence of a covalent bond. A similar band is observed in high-resolution electron energy loss spectra of nitrobenzene layers on planar gold. The bonding of these types of films through a covalent interaction on gold is consistent with their enhanced stability observed in other studies. These findings provide motivation for the use of diazonium-derived films on gold and other metals in applications where high stability and/or strong adsorbate-substrate coupling are required.

An enzyme-linked assay for the rapid quantification of microRNAs based on the viral suppressor of RNA silencing protein p19.

MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19's utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1fmol and a linear dynamic range from 1pmol to 1fmol.