Label-Free Multimodal Protease Detection Based on Protein/Perylene Dye Coassembly and Enzyme-Triggered Disassembly

The development of novel assays for protease sensing plays an important role in clinical diagnostics and therapeutics. Herein, we report a supramolecular platform for label-free protease detection, based on protein/dye self-assembly and enzyme-triggered disassembly. In a typical case, coassembly of protamine sulfate and perylene dye via electrostatic attractions and π–π interactions caused significant colorimetric and fluorescent responses. Subsequent addition of trypsin was found to cleave the amide bonds of protein, triggering the dissociation of protein/dye aggregates and the release of perylene dyes. The enzyme-triggered disassembly was transduced into multiple readouts including absorption, fluorescence, and polarization, which were exploited for trypsin detection and inhibitor testing. This assay was also used for turn-on fluorescence detection of cathepsin B, an enzyme known to be overexpressed in mammalian cancer cells. The integration of supramolecular self-assembly into enzyme detection in this work has provided a novel label-free biosensing platform which is highly sensitive with multimodal readouts. The relative simplicity of the approach avoids the need for time-consuming substrate synthesis, and is also amenable to naked eye detection

Polymerization Amplified Detection for Nanoparticle-Based Biosensing

Efficient signal amplification processes are key to the design of sensitive assays for biomolecule detection. Here, we describe a new assay platform that takes advantage of both polymerization reactions and the aggregation of nanoparticles to amplify signal. In our design, a cascade is set up in which radicals generated by either enzymes or metal ions are polymerized to form polymers that can entangle multiple gold nanoparticles (AuNPs) into aggregates, resulting in a visible color change. Less than 0.05% monomer-to-polymer conversion is required to initiate aggregation, providing high sensitivity toward the radical generating species. Good sensitivity of this assay toward horseradish peroxidase, catalase, and parts per billion concentrations of iron and copper is shown. Incorporation of the oxygen-consuming enzyme glucose oxidase (GOx), enables this assay to be performed in open air conditions at ambient temperature. We anticipate that such a design will provide a useful platform for sensitive detection of a broad range of biomolecules through polymerization-based amplification

Highly Controlled Open Vessel RAFT Polymerizations by Enzyme Degassing

Intolerance to oxygen is a key limitation in many reactions and particularly in controlled radical polymerizations. Here we introduce the use of enzymes such as glucose oxidase (GOx) to deoxygenate reversible addition–fragmentation chain transfer polymerizations (Enz-RAFT), facilitating the preparation of highly controlled polymers in vessels open to ambient oxygen. Because the removal of oxygen is so efficient, very low concentrations of GOx and initiator can be used, enabling excellent control which is demonstrated by pseudoliving polymerization kinetics and the preparation of multiblock copolymers with narrow molecular weight distributions (<i>M</i>_w/<i>M</i>_n < 1.15). GOx retains sufficient activity to facilitate polymerization not only in aqueous solutions but also in a range of water/organic solvent mixtures, and we demonstrate the use of this technique to perform open vessel Enz-RAFT polymerizations in various methanol and dioxane/water mixtures

Swollen Micelles for the Preparation of Gated, Squeezable, pH-Responsive Drug Carriers

Natural variations in pH levels of tissues in the body make it an attractive stimuli to trigger drug release from a delivery vehicle. A number of such carriers have been developed but achieving high drug loading combined with low leakage at physiological pH and tunable controlled release at the site of action is an ongoing challenge. Here we report a novel strategy for the synthesis of entirely hydrophilic stimuli-responsive nanocarriers with high passive loading efficiency of doxorubicin (DOX), which show good stability at pH 7 and rapid tunable drug release at intracellular pH. The particles (<i>D</i>_h = 120–150 nm), are prepared by cross-linking the core of swollen micelles of the triblock copolymer poly­[poly­(ethylene glycol) methyl ether methacrylate-<i>b</i>-<i>N</i>,<i>N</i>′-di(methylamino)ethyl methacrylate-<i>b</i>-<i>tert</i>-butyl methacrylate] (poly­(PEGMEM A)<i>-<i>b</i>-</i> PDMAEMA-<i>b</i>-P<i>t</i>BMA)). After subsequent deprotection of the <i>tert</i>-butyl groups a hydrophilic poly­(methacrylic acid) (PMAA) core is revealed. Due to the negative charge in the acidic core the particles absorb 100% of the DOX from solution at pH 7 at up to 50 wt % DOX/polymer, making them extremely simple to load. Unlike other systems, the DMAEMA “gating” shell ensures low drug leakage at pH 7, whereas physical shrinkage of the MAA core allows rapid release below pH 6. The particles deliver DOX with high efficiency to human pancreatic cancer AsPC-1 cell lines, even lowering the IC50 of DOX. As the particles are stable as a dry powder and can be loaded with any mixture of positively charged drugs without complex synthetic or purification steps, we propose they will find use in a range of delivery applications

Consensus too soon: judges’ and lawyers’ views on genetic information use

Timely effective regulation of genetic advances presents a challenge for justice systems. We used a 51-item battery to examine views on major genetics-related issues of those at the forefront of regulating this area – Supreme Court judges (N = 73). We also compared their views with those of other justice stakeholders (N = 210) from the same country (Romania). Judges showed greater endorsement and less variability in views on the use of genetic data and technologies than the other groups. The agreement among the judges was strikingly strong for some controversial issues, including gene editing; patenting of genetic findings; and the State using genetic information for crime prevention. Judges and other lawyers recognized the need for amending the relevant laws. Without appropriate regulation, genetic science has a risk of propelling inequality rather than fulfilling its promise to improve people’s lives.</p

All-Optical Thermometry and Thermal Properties of the Optically Detected Spin Resonances of the NV^– Center in Nanodiamond

The negatively charged nitrogen-vacancy (NV^–) center in diamond is at the frontier of quantum nanometrology and biosensing. Recent attention has focused on the application of high-sensitivity thermometry using the spin resonances of NV^– centers in nanodiamond to subcellular biological and biomedical research. Here, we report a comprehensive investigation of the thermal properties of the center’s spin resonances and demonstrate an alternate all-optical NV^– thermometry technique that exploits the temperature dependence of the center’s optical Debye–Waller factor

Polymeric Nanocapsules for Enzyme Stabilization in Organic Solvents

Herein we report an approach to encapsulate enzymes within polymeric nanocapsules dispersed in an organic solvent via inverse miniemulsion periphery RAFT polymerization (IMEPP). Glucose oxidase (GOx), which has various applications but is unstable at elevated temperature and in organic solvents, was chosen as a model enzyme. In this study, we have explored the use of photoinitiation under visible (blue) light instead of thermal initiation to avoid enzyme denaturation by heating. GOx was successfully encapsulated within polymeric nanocapsules (∼200 nm) and showed high activity (71–100% relative to free GOx in PBS) dispersed in toluene/<i>t</i>-BuOH. The nanocapsules were thus able to protect GOx and enable it to function in an organic solvent mixture where native GOx would otherwise undergo denaturation. This approach of enzyme encapsulation is significant as it may lead to increased industrial applications of enzyme biocatalysis, expanding the use of enzymes as nontoxic and environmentally friendly biocompatible catalysts

Soft gamma repeaters and short gamma ray bursts: Making magnetars from WD-WD mergers

Recent progress on the nature of short duration Gamma Ray Bursts (GRBs) has shown that a fraction of them originate in the local universe. These systems may well be the result of giant flares from Soft Gamma Repeaters (SGRs) believed to be magnetars (neutron stars with extremely large magnetic fields ⩾10^14 G). If these magnetars are formed via the core collapse of massive stars, then it would be expected that the bursts should originate from predominantly young stellar populations. However, correlating the positions of BATSE short bursts with structure in the local universe reveals a correlation with all galaxy types, including those with little or no ongoing star formation. This is a natural outcome if, in addition to magnetars forming via the core collapse of massive stars, they also form via Accretion Induced Collapse following the merger of two white dwarfs, one of which is magnetic. We investigate this possibility and find that the rate of magnetar production via WD‐WD mergers in the Milky Way is comparable to the rate of production via core collapse. However, while the rate of magnetar production by core collapse is proportional to the star formation rate, the rate of production via WD‐WD mergers (which have long lifetimes) is proportional to the stellar mass density, which is concentrated in early‐type systems. Therefore magnetars produced via WD‐WD mergers may produce SGR giant flares which can be identified with early type galaxies. We also comment on the possibility that this mechanism could produce a fraction of the observed short duration GRB population at low redshift

Genes differentially expressed in short survival versus long survival patients, considered according to cancer type and cancer class.

Volcano plots showing significantly downregulated (the thresholds are FDR -0.5) genes in green and red, respectively. Here short survival patients are compared with long survival patients, so in all three panels genes found to be upregulated are genes that are upregulated in short survival patients, and genes found downregulated are genes that are downregulated in short survival patients. Grey indicates genes that are not significantly downregulated or upregulated. Log of fold change (LogFC) is on the x-axis and significance level (-log10P) is on the y-axis. Panel A shows differentially expressed genes when all of the patients are considered (n = 515). Panel B shows differentially expressed genes when only patients with solid cancers are considered (n = 293), and panel C shows differentially expressed genes when only patients with blood cancers are considered (n = 222). In all panels, the significantly downregulated and upregulated genes are labelled with their Hugo Gene Nomenclature Committee (HGNC) gene symbols. The three genes (SSX1, MAGEC2 and ULBP2) that are found to be significantly differentially expressed in all three analyses are shown in bold.</p

Patients statistics.

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