Minimal Substrate Research Articles

Rapid on-site colorimetric detection of Arsenic(V) by NH2-MIL-88(Fe) nanozymes-based Ultraviolet-visible spectroscopic and smartphone-assisted sensing platforms

BackgroundBecause arsenate (As(V)) is a highly toxic pollutant, timely on-site monitoring of its concentration is crucial for mitigating potential environmental and health hazards. Traditional on-site detection methods for As(V) often face limitations of long response time and low sensitivity. Nanozymes are nanomaterials that exhibit enzyme-like catalytic activity. Nanozyme-based colorimetric detection can amplify signals and improve detection sensitivity by catalytically converting a minimal substrate quantity into a substantial amount. However, current nanozymes-based As(V) detection methods still suffer from prolonged response time and the lack of convenient detection tools. ResultsWe develop a rapid and sensitive strategy for on-site colorimetric As(V) detection using a metal-organic framework (MOF) nanozyme, NH2-MIL-88(Fe). NH2-MIL-88(Fe) featured abundant Fe unsaturated metal centers (UMCs) and ordered porous structure, exhibiting excellent peroxidase-like activity, rapid As(V) adsorption, and high water dispersity. Fe UMCs efficiently catalyzed the oxidization of colorless tetramethylbenzidine (TMB) to blue oxTMB in the presence of H2O2. As(V) selectively inhibited this activity, reducing Ultraviolet-visible (UV-vis) absorption at 650 nm and fading the solution color. Mechanistically, As(V) interacted with Fe UMCs through As-O-Fe bonds, impeding Fe3+ reduction and Fe2+ catalytic ability, reducing •OH production. Under optimized conditions, As(V) was detected within 15 minutes, with detection limits of 2.78 μg•L-1 via UV-vis and 13.56 μg•L-1 via smartphone-assisted platform, covering a linear range of 5.00 - 600.00 μg•L-1. Additionally, NH2-MIL-88(Fe) was incorporated into an agarose hydrogel to create a portable composite for smartphone-based colorimetric analysis As(V). Significance and noveltyThis study addresses the existing issues of nanozyme-based As(V) sensors, elucidates the molecular mechanism by which As(V) affects NH2-MIL-88(Fe) nanozymes activity, and confirms the precision and accuracy of the established method in spiked river samples. Our rapid, sensitive, and facile approach offers a practical and efficient solution for on-site As(V) detection, facilitating swift intelligent risk identification and effective pollution prevention and remediation.

Modeling, simulations, and Simulink developments in the analysis of optimal control of temperature and pH in a batch ethanol fermentation process

Transfer of laboratory-scale experiments to production-scale ethanol fermentation is time-consuming and involves expensive prototype systems from complex experimental designs that determine optimal operating conditions for minimal substrate and product inhibitions. The study developed and validated a Simulink-based model for optimal pH and temperature control using fuzzy logic and PID controllers respectively and taking advantage of 2D and 1D substrate and product inhibition models from which suitable ethanol fermentation reaction rates models were selected. Temperature and pH levels and substrate, product, and biomass concentrations were measured. Selected inhibition models were linear-product, linear substrate-sudden stop product, and linear substrate for cassava, maize, and sorghum, respectively. Fuzzy logic controller ascertained optimal flow rate of acid and base as 0.000196 ml/s and 0.000204 ml/s, respectively, and pH error and rate of pH error as 0.00334 and 0.00368, respectively. F-test two-sample for variances showed no significant difference between model and experimental curves (cassava: F critical = 0.9704, F calculated = 0.1905; maize: F critical 0.9704, F calculated = 0.2149; sorghum: F critical = 0.9704, F calculated = 0.2488). PID logic controller showed model curves and experimental curves with good fit. F-test two-sample for variances showed no significant difference between model and experimental curves (cassava: F critical = 0.9704, F calculated = 0.1288; maize: F critical = 0.9704, F calculated = 0.2083; sorghum: F critical = 0.9704, F calculated = 0.2016). The study provided an improved approach as solution for optimal pH and temperature conditions in order to mitigate substrate and product inhibitions during ethanol fermentation. It illustrated that the application of artificial intelligence-based controllers provides satisfactory outcomes that are desirable for implementation in the industrial space.Graphical

Degradation of Natural Undaria pinnatifida into Unsaturated Guluronic Acid Oligosaccharides by a Single Alginate Lyase.

Here, we report on a bifunctional alginate lyase (Vnalg7) expressed in Pichia pastoris, which can degrade natural Undaria pinnatifida into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture) was 15.81-fold higher than that of the original alg (228.90 U/mL-culture), following engineering modification. The degradation rate reached 52.75%, and reducing sugar reached 30.30 mg/mL after combining Vnalg7 (200.00 U/mL-culture) and 14% (w/v) U. pinnatifida for 6 h. Analysis of the action mode indicated that Vnalg7 could degrade many substrates to produce a variety of unsaturated alginate oligosaccharides (AOSs), and the minimal substrate was tetrasaccharide. Site-directed mutagenesis showed that Glu238, Glu241, Glu312, Arg236, His307, Lys414, and Tyr418 are essential catalytic sites, while Glu334, Glu344, and Asp311 play auxiliary roles. Mechanism analysis revealed the enzymatic degradation pattern of Vnalg7, which mainly recognizes and attacks the third glycosidic linkage from the reducing end of oligosaccharide substrate. Our findings provide a novel alginate lyase tool and a sustainable and commercial production strategy for value-added biomolecules using seaweeds.

The minimal computational substrate of fluid intelligence

The quantification of cognitive powers rests on identifying a behavioural task that depends on them. Such dependence cannot be assured, for the powers a task invokes cannot be experimentally controlled or constrained a priori, resulting in unknown vulnerability to failure of specificity and generalisability. Evaluating a compact version of Raven's Advanced Progressive Matrices (RAPM), a widely used clinical test of fluid intelligence, we show that LaMa, a self-supervised artificial neural network trained solely on the completion of partially masked images of natural environmental scenes, achieves representative human-level test scores a prima vista, without any task-specific inductive bias or training. Compared with cohorts of healthy and focally lesioned participants, LaMa exhibits human-like variation with item difficulty, and produces errors characteristic of right frontal lobe damage under degradation of its ability to integrate global spatial patterns. LaMa's narrow training and limited capacity suggest matrix-style tests may be open to computationally simple solutions that need not necessarily invoke the substrates of reasoning.

Novel deuterated cyclopentadienyl zirconium/hafnium precursors for atomic layer deposition of high-performance ZrO2/HfO2 thin films

The need to improve the electrical properties of ZrO2 and HfO2 thin films deposited by atomic layer deposition (ALD), which is widely used in the field of microelectronics, is growing. Attempts to improve precursor stability unavoidably lead to reduced reactivity and diminished utility. Therefore, we synthesized and developed two new ALD precursors by introducing deuterium, which simultaneously exhibit enhanced thermal stability and enhanced reactivity as a result of the introduction of deuterium. For the substitution of deuterium for hydrogen in the precursors responsible for the enhanced thermal stability, we experimentally and theoretically demonstrated that thermal stability and reactivity can be simultaneously enhanced by reducing the bonding dissociation energy of ligands that have not undergone deuterium substitution. This observation led to the development of ZrO2 and HfO2 ALD processes that result in no impurities within the thin films and minimal substrate damage even at a very high deposition temperature of 400 °C. In particular, the improvement in crystallinity through ultra-high-temperature ALD deposition resulted in excellent electrical properties in a metal–insulator–metal capacitor in which the film was incorporated; the capacitor demonstrated a dielectric constant of 37.9 and leakage current of 3.52 × 10−9 A cm−2, without the need for a subsequent annealing process.

Realization of multi-functional features with ZnO nanosheets/p-Si based electronic device for energy harvesting and memristive switching

This work investigates and reports on the fabrication of a ZnO nanosheets/p-Si heterojunction energy harvester. The proposed nanostructure device exhibits two key functionalities: energy harvesting and memristive characteristics. This allows the device to perform multiple tasks. The ZnO nanostructure sheet was grown using a hydrothermal method. To minimize defect states at the electrode-substrate interface, an optimal phosphorus doping process was employed to achieve minimal substrate sheet resistance. Under an applied pushing force of 0.259 kgf, the energy harvester generated an output voltage and current of 0.5548 V and 44 μA, respectively. The proposed structure produces an output of 24.41 μW at 13 Hz for 2000 cycles. Investigation of the device's transfer characteristics revealed memristive behavior with an on/off ratio of 107. These findings suggest that the multifunctional ZnO nanosheets/p-Si electronic device reported here has promising potential for applications in the Internet of Things (IoT).

Reassignment of the Structure of a Tryptophan-Containing Cyclic Tripeptide Produced by the Biarylitide Crosslinking Cytochrome P450blt.

The structure of the sidechain crosslinked Tyr-Leu-Trp peptide produced by the biarylitide crosslinking cytochrome P450Blt from Micromonospora sp. MW-13 has been reanalysed by a series of NMR, computational and isotope labelling experiments and shown to contain a C-N rather than a C-O bond. Additional in vivo experiments using such a modified peptide show there is a general tolerance of biarylitide crosslinking P450 enzymes for histidine to tryptophan mutations within their minimal peptide substrate sequences despite the lack of such residues noted in natural biarylitide gene clusters. This work further highlights the impressive ability of P450s from biarylitide biosynthesis pathways to act as biocatalysts for the formation of a range of sidechain crosslinked tripeptides.

MutL Activates UvrD by Interaction Between the MutL C-terminal Domain and the UvrD 2B Domain

UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3′-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.

A minimal RNA substrate with dual fluorescent probes enables rapid kinetics and provides insight into bacterial RNase P active site interactions.

Bacterial ribonuclease P (RNase P) is a tRNA processing endonuclease that occurs primarily as a ribonucleoprotein with a catalytic RNA subunit (P RNA). As one of the first ribozymes discovered, P RNA is a well-studied model system for understanding RNA catalysis and substrate recognition. Extensive structural and biochemical studies have revealed the structure of RNase P bound to precursor tRNA (ptRNA) and product tRNA. These studies also helped to define active site residues and propose the molecular interactions that are involved in substrate binding and catalysis. However, a detailed quantitative model of the reaction cycle that includes the structures of intermediates and the process of positioning active site metal ions for catalysis is lacking. To further this goal, we used a chemically modified minimal RNA duplex substrate (MD1) to establish a kinetic framework for measuring the functional effects of P RNA active site mutations. Substitution of U69, a critical nucleotide involved in active site Mg2+ binding, was found to reduce catalysis >500-fold as expected, but had no measurable effect on ptRNA binding kinetics. In contrast, the same U69 mutations had little effect on catalysis in Ca2+ compared to reactions containing native Mg2+ ions. CryoEM structures and SHAPE mapping suggested increased flexibility of U69 and adjacent nucleotides in Ca2+ compared to Mg2+. These results support a model in which slow catalysis in Ca2+ is due to inability to engage U69. These studies establish a set of experimental tools to analyze RNase P kinetics and mechanism and can be expanded to gain new insights into the assembly of the active RNase P-ptRNA complex.

Structure-based mining of a chitosanase with distinctive degradation mode and product specificity.

Chitosan derivates with varying degrees of polymerization (DP) have attracted great concern due to their excellent biological activities. Increasing the abundance of chitosanases with different degradation modes contributes to revealing their catalytic mechanisms and facilitating the production of chitosan derivates. However, the identification of endo-chitosanases capable of producing chitobiose and D-glucosamine (GlcN) from chitosan substrates has remained elusive. Herein, an endo-chitosanase (CsnCA) belonging to the GH46 family was identified based on structural analysis in phylogenetic evolution. Moreover, we demonstrate that CsnCA acts in a random endo-acting manner, producing chitosan derivatives with DP ≤ 2. The in-depth analysis of CsnCA revealed that (GlcN)3 serves as the minimal substrate, undergoing cleavage in the mode that occupies the subsites - 2 to + 1, resulting in the release of GlcN. This study succeeded in discovering a chitosanase with distinctive degradation modes, which could facilitate the mechanistic understanding of chitosanases, further empowering the production of chitosan derivates with specific DP. KEY POINTS: • Structural docking and evolutionary analysis guide to mining the chitosanase. • The endo-chitosanase exhibits a unique GlcN-producing cleavage pattern. • The cleavage direction of chitosanase to produce GlcN was identified.

A Microfluidic Countercurrent Reactor for Accelerating Enzymatic Reactions

AbstractEnzymes are important catalysts in biochemical reactions with superior regio, stereo, and substrate selectivity. However, enzymatic reaction systems have drawbacks including product inhibition, difficulty recycling, and poor stability. Importantly, the rate of an enzyme catalyzed reaction diminishes rapidly due to product inhibition and substrate depletion, making it difficult for many enzymes to catalyze a reaction to completion. The outcome is a mixture of unreacted substrates being present in the final reaction, necessitating additional separation steps that increase costs. This study presents a microfluidic reactor for accelerating enzyme catalyzed reactions using a countercurrent design that continuously removes products and adds fresh substrate into the reaction, allowing enzymes to operate under better reaction conditions. It demonstrates that countercurrent flow accelerates enzymatic reactions in our system up to 36 % for horseradish peroxidase and 21 % for β‐glucosidase compared to cocurrent flow, and the resulting reaction solution contains highly pure product with minimal substrate contamination.

A novel alginate lyase and its domain functions for the preparation of unsaturated monosaccharides.

Brown algae are considered promising crops for the production of sustainable biofuels. However, the commercial application has been limited by lack of efficient methods for converting alginate into fermentable sugars. Herein, we cloned and characterized a novel alginate lyase AlyPL17 from Pedobacter hainanensis NJ-02. It possessed outstanding catalytic efficiency toward polymannuronic acid (polyM), polyguluronic acid (polyG), and alginate sodium, with kcat of 39.42 ± 1.9s-1, 32.53 ± 0.88s-1, and 38.30 ± 2.12s-1, respectively. AlyPL17 showed maximum activity at 45°C and pH 9.0. The domain truncation did not change the optimal temperature and optimal pH, but greatly reduced the activity. In addition, AlyPL17 degrades alginate through the cooperative action of two structural domains in an exolytic mode. The minimal degradation substrate of AlyPL17 is a disaccharide. Furthermore, AlyPL17 and AlyPL6 can synergistically degrade alginate to prepare unsaturated monosaccharides that can be converted to 4-deoxy-L-erythron-5-hexoseuloseuronate acid (DEH). DEH is reduced to KDG by DEH reductase (Sdr), which enters the Entner-Doudoroff (ED) pathway as a common metabolite and is converted to bioethanol. KEY POINTS: • Biochemical characterization of alginate lyase from Pedobacter hainanensis NJ-02 and its truncated form. • Degradation patterns of AlyPL17 and the role of its domains in product distribution and mode of action. • Potential of synergistic degradation system for efficient preparation of unsaturated monosaccharides.

Characterization of degradation patterns and enzymatic properties of a novel alkali-resistant alginate lyase AlyRm1 from Rubrivirgamarina

Alginate lyase is essential for the production of alginate oligosaccharides (AOSs), which exhibit diverse bioactivities and have numerous applications in the food and pharmaceutical industries. The creation of recombinant alginate lyase by genetic engineering lays a crucial foundation for the commercialization of alginate lyase. This study cloned and expressed the polysaccharide lyase family 6 (PL6) alginate lyase gene alyrm1 from Rubrivirga marina.The optimum temperature and pH for recombinant AlyRm1 are 30 °C and 10.0, respectively. AlyRm1 shows good alkaline stability, for it remained over 80% of the enzyme activity after being incubated at pH 10.0 for 24 h AlyRm1 preferentially degrades PolyM into AOSs with degrees of polymerization (DP) 2–5 and monosaccharides as an endolytic bifunctional lyase. In addition, the analysis of degradation products toward oligosaccharides revealed that the minimal substrate of AlyRm1 is trisaccharide and clarified the degradation patterns.

Structure of the Reductase Domain of a Fungal Carboxylic Acid Reductase and Its Substrate Scope in Thioester and Aldehyde Reduction.

The synthesis of aldehydes from carboxylic acids has long been a challenge in chemistry. In contrast to the harsh chemically driven reduction, enzymes such as carboxylic acid reductases (CARs) are considered appealing biocatalysts for aldehyde production. Although structures of single- and didomains of microbial CARs have been reported, to date no full-length protein structure has been elucidated. In this study, we aimed to obtain structural and functional information regarding the reductase (R) domain of a CAR from the fungus Neurospora crassa (Nc). The NcCAR R-domain revealed activity for N-acetylcysteamine thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate and can be anticipated as the minimal substrate for thioester reduction by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate, which is in good agreement with docking experiments performed with the minimal substrate. In vitro studies were performed with this highly purified R-domain and NADPH, demonstrating carbonyl reduction activity. The R-domain was able to accept not only a simple aromatic ketone but also benzaldehyde and octanal, which are typically considered to be the final product of carboxylic acid reduction by CAR. Also, the full-length NcCAR reduced aldehydes to primary alcohols. In conclusion, aldehyde overreduction can no longer be attributed exclusively to the host background.

Assay for ADAMTS-13 Activity with Flow Cytometric Readout.

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) is a metalloprotease that regulates the size of circulating von Willebrand factor (vWF) multimers. Severe lack of ADAMTS-13 activity [<10% of normal (0.1 IU/mL)] leads to thrombotic thrombocytopenic purpura (TTP), a specific type of thrombotic microangiopathy (TMA). Timely determination of plasma ADAMTS-13 activity is essential to discriminate TTP from other types of TMA with respect to adequate treatment. Identification of the minimal substrate motif for ADAMTS-13 within the A2 domain of vWF (vWF73) as well as the generation of monoclonal antibodies (mAbs) that specifically recognize the ADAMTS-13 cleavage site enabled the development of a variety of methods for determination of plasma ADAMTS-13 activity. In order to further extend the range of analytical platforms applicable for quantitative determination of plasma ADAMTS-13 activity, a specific, vWF/mAb-based assay with flow cytometric readout was developed and validated. Basic assay characteristics include a total assay time of 80 to 90 min, a near linear dynamic range from 0.005 (lower limit of quantification) to 0.2 IU/mL, and intra- and interassay coefficients of variation below 5 and 30% at input plasma ADAMTS-13 activities of 0.015 and ≤0.050 IU/mL, respectively. When compared to the results obtained with a commercially available quantitative ADAMTS-13 activity ELISA, analysis of 18 plasma samples obtained from patients with suspected TTP revealed full agreement of results with respect to the clinical 0.1 IU/mL TTP threshold. Based on these data, it is assumed that the described assay principle can be successfully transferred to virtually all laboratories that have a flow cytometer available.

Leveraging Substrate Promiscuity of a Radical S-Adenosyl-L-methionine RiPP Maturase toward Intramolecular Peptide Cross-Linking Applications.

Radical S-adenosyl-l-methionine (RS) enzymes operate on a variety of substrates and catalyze a wide range of complex radical-mediated transformations. Radical non-α-carbon thioether peptides (ranthipeptides) are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs). The RS enzyme PapB catalyzes the formation of thioether cross-links between Cys/Asp (or Cys/Glu) residues located in six Cys-X3-Asp/Glu motifs. In this report, using a minimal substrate that contains a single cross-link motif, we explore the substrate scope of the PapB and show that the enzyme is highly promiscuous and will accept a variety of Cys-Xn-Asp sequences where n = 0–6. Moreover, we show that the enzyme will introduce in-line and nested thioether cross-links independently in peptide sequences that contain two motifs derived from the wild-type sequence. Additionally, the enzyme accepts peptides that contain d-amino acids at either the Cys or the Asp position. These observations are leveraged to produce a thioether cyclized analogue of the FDA-approved therapeutic agent octreotide, with a Cys-Glu cross-link replacing the disulfide that is found in the drug. These findings highlight the remarkable substrate tolerance of PapB and show the utility of RS RiPP maturases in biotechnological applications.

Investigation and Optimization of Process Parameters of Abrasive Water Jet Cutting on C360 Brass Alloy through Design of Experiments

Many scientists concentrate on features of water jet cutting by researching various input parameters, abrasive mesh sizes to improve improved processing conditions without the expense. In this work, the treatment of abrasive water jet should be planned to ensure minimal surface roughness and maximum substrate removal. C360 brass alloy is considered to optimize the parameters for cutting including the abrasive feed, stand-off distance and nozzle speeds via the design of experiments supported with response surface methodology.

Abstract 5076: Evaluation of the CNS penetration of a next generation PARP inhibitor, AZD9574, in cynomolgus monkey using positron emission tomography

Abstract The current clinically approved PARP inhibitors have limited subtype selectivity and are to some degree restricted in their ability to penetrate the central nervous system (CNS) due to efflux transporters, potentially limiting their efficacy in treating metastatic disease or primary tumors in the brain. The current study evaluated the potential of AZD9574, a next generation, PARP1 selective inhibitor/trapper, to penetrate the CNS in cynomolgus monkey, and its occupancy of the PARP1 enzyme, using positron emission tomography (PET). In vitro bidirectional efflux assay data suggested AZD9574 showed minimal substrate potential compared to the clinically approved PARP inhibitors. This was reflected in an increased ratio of unbound brain to unbound plasma concentration (Kpuu) in the rat of ~0.31. Therefore AZD9574 was taken forward into cynomolgus monkey PET studies. Firstly, the Kpuu was determined following dosing of [11C]AZD9574, co-administered with unlabeled drug to minimize the impact of specific binding. The high specific signal observed lead to the development of [11C]AZ3391, a PARP1 selective, CNS penetrant PET tracer, which was subsequently used to directly assess the PARP1 target engagement of AZD9574 in the brain. AZD9574 was found to show a Kpuu in cynomolgus monkeys of 0.79, close to unity with unbound plasma concentrations suggesting minimal CNS restriction. Furthermore, an i.v. infusion dose response study with AZD9574, conducted to examine its ability to block target occupancy by the PET tracer [11C]AZ3391, demonstrated a reduction in [11C]AZ3391 accumulation in whole brain. The resulting calculated occupancy of AZD9574 ranged from 17% for the lowest dose (0.003 mg/kg) to 95% for the highest dose tested (0.05 mg/kg). Comparable reduction in occupancy was seen for peripheral tissue, such as bone marrow, supporting the conclusion that AZD9574 shows minimal CNS restriction. These data show that AZD9574 is the first PARP inhibitor to reach the clinic which combines PARP1 selectivity, trapping and high CNS penetration in a single molecule and supports its development as a potential therapy for the treatment of metastatic disease and primary brain tumors. Citation Format: Andy Pike, Amber Balazs, Zsolt Cselényi, Sébastien L. Degorce, Avipsa Ghosh, Sudhir M. Hande, Jeffrey Johannes, Peter Johnström, Martin J. Packer, Magnus Schou, XiaoLan Zheng. Evaluation of the CNS penetration of a next generation PARP inhibitor, AZD9574, in cynomolgus monkey using positron emission tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5076.

Fluorescence-Based Activity Screening Assay Reveals Small Molecule Inhibitors of Vaccinia Virus mRNA Decapping Enzyme D9.

Vaccinia virus (VACV) represents a family of poxviruses, which possess their own decapping machinery as a part of their strategy to eliminate host mRNAs and evade the innate immune response. D9 is one of the two encoded VACV decapping enzymes that is responsible for cap removal from the 5′ end of both host mRNA transcripts and viral double-stranded RNAs. Little is known about the structural requirements for D9 inhibition by small molecules. Here, we identified a minimal D9 substrate and used it to develop a real-time fluorescence assay for inhibitor discovery and characterization. We screened a panel of nucleotide-derived substrate analogues and pharmacologically active candidates to identify several compounds with nano- and low micromolar IC50 values. m7GpppCH2p was the most potent nucleotide inhibitor (IC50 ∼ 0.08 μM), and seliciclib and CP-100356 were the most potent drug-like compounds (IC50 0.57 and 2.7 μM, respectively). The hits identified through screening inhibited D9-catalyzed decapping of 26 nt RNA substrates but were not active toward VACV D10 or human decapping enzyme, Dcp1/2. The inhibition mode for one of the compounds (CP-100356) was elucidated based on the X-ray cocrystal structure, opening the possibility for structure-based design of novel D9 inhibitors and binding probes.

Electron transport in discontinuous metal thin films

The structure and basic experimental electrical properties of vacuum evaporated discontinuous (island) metal thin films of discrete metal nanoparticles on insulating substrates are briefly reviewed. Then the widely accepted Neugebauer and Webb (N&W) electrostatically activated electron tunneling conduction model is covered (with enhancements) before the numerous discrepancies between this model and experimental observations are identified, e.g. minimal substrate bias effect, non-linear field distribution, anomalous AC effects, asymmetrical contact effects, and switching. A modified model, based on contact electron injection and extraction, and computer simulations are introduced which explain these discrepancies at a qualitative level. However, quantitative experimental verification of the model is not possible without stable, reproducible films of known structures. The paper concludes with a review of possible preparation techniques which could yield satisfactory samples, especially self-assembly of organically protected metal nanoparticles. One of these has already demonstrated electrostatically activated conduction.