Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development ...and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.
Spirocyclic oxindoles undergo an enantioselective oxygenation reaction (nine examples; e.r. up to 97:3) upon catalysis by a chiral ruthenium porphyrin complex (1 mol %). The catalyst exhibits a ...lactam ring, which is responsible for substrate association through hydrogen bonds, and an active ruthenium center, which is in a defined spatial relationship to the oxygenation substrate. DFT calculations illustrate the perfect alignment of the active site with the reactive CH bond and suggest—in line with the kinetic isotope effect—an oxygen rebound mechanism for the reaction.
Correctly addressed: Catalyst 1 allows for a selective oxygenation of spirocyclic oxindoles at one of two enantiotopic positions (
). Evidence was collected that hydrogen bonding (
) is responsible for a perfect spatial overlap of the reactive centers in the transition state of the CH activation reaction.
An iridium catalyzed method for the synthesis of functionalized cyclohexanes from methyl ketones and 1,5-diols is described. This process operates by two sequential hydrogen borrowing reactions, ...providing direct access to multisubstituted cyclic products with high levels of stereocontrol. This methodology represents a novel (5 + 1) strategy for the stereoselective construction of the cyclohexane core.
The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms that may play an important role in recurrent C. difficile infections. The mechanisms underlying C. difficile community ...formation and inter-bacterial interactions are nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT) and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with other gut species, C. difficile interactions with a common gut bacterium, Bacteroides fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C. difficile LuxS alleviates the B. fragilis-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.
The application of an iridium-catalyzed hydrogen borrowing process to enable the formation of α-branched ketones with higher alcohols is described. In order to facilitate this reaction, ...ortho-disubstituted phenyl and cyclopropyl ketones were recognized as crucial structural motifs for C–C bond formation. Having optimized the key catalysis step, the ortho-disubstituted phenyl products could be further manipulated by a retro-Friedel–Crafts acylation reaction to produce synthetically useful carboxylic acid derivatives. In contrast, the cyclopropyl ketones underwent homoconjugate addition with several nucleophiles to provide further functionalized branched ketone products.
Aims:
Encouraging planting in front gardens offers mental and physical health benefits, as well as positive local environmental impacts such as reducing flood risk and improving air quality. However, ...urban front garden greenery has reduced in recent years. We aimed to explore adults’ views regarding planting greenery in front gardens, barriers and facilitators, and their understanding of health and environmental impacts, to identify appropriate intervention mechanisms for behaviour change.
Methods:
We carried out five online focus groups with 20 participants aged 20–64 in England, purposively sampled for variation according to age, gender, home ownership, income, ethnicity and residing in an urban or suburban area. We audio recorded each focus group, transcribed it verbatim and analysed transcripts using thematic analysis.
Results:
Front gardening was a relaxing activity that provided benefits including increased wellbeing, fresh air and vitamin D. Planting in front gardens depended heavily on available time and space, garden orientation, local security and the weather. Front gardens could be a place for social interaction. Participants tended to prioritise neatness and tidiness over greenery. Lack of knowledge and low self-efficacy were key barriers. There was little awareness of the environmental benefits of front garden greenery; however, reducing flood risk and encouraging biodiversity were viewed positively.
Conclusion:
Initiatives to encourage front garden planting should focus on plants that require little knowledge to acquire and care for, are suitable to the local environmental conditions and with a visual impact of neatness and bright colour. Campaigns should draw attention to local flood risk reduction and increasing biodiversity, in addition to personal health benefits.
Knowledge of the neutron light-yield response is crucial to the understanding of scintillator-based neutron detectors. In this work, neutrons from 2–6MeV have been used to study the scintillation ...light-yield response of the liquid scintillators NE 213A, EJ 305, EJ 331 and EJ 321P using event-by-event waveform digitization. Energy calibration was performed using a GEANT4 model to locate the edge positions of the Compton distributions produced by gamma-ray sources. The simulated light yield for neutrons from a PuBe source was compared to measured recoil proton distributions, where neutron energy was selected by time-of-flight. This resulted in an energy-dependent Birks parameterization to characterize the non-linear response to the lower energy neutrons. The NE 213A and EJ 305 results agree very well with existing data and are reproduced nicely by the simulation. New results for EJ 331 and EJ 321P, where the simulation also reproduces the data well, are presented.
Choline feeding in the form of rumen-protected choline (RPC) has been shown to increase milk production and improve measures of metabolic health (e.g., liver triglyceride) in dairy cows. The ...objective was to characterize changes in plasma and milk choline and choline metabolite concentrations, including microbial-derived trimethylamine N-oxide (TMAO), in response to increasing ruminal spot-doses, different types of RPC, and ruminal stability of RPC in lactating cows. For experiment 1, 12 mid-lactation (121 ± 16.3 d in milk) Holstein cows were balanced by total plasma choline concentrations and milk yields. Cows were assigned to 1 of 3 lipid-encapsulated RPC products (main plots): prototypes P1, P2, and P3 (containing 59, 56, and 30% choline chloride, respectively). Within each main plot, cows were assigned to a sequence of doses in a 4 × 4 Latin square design: 0, 18, 36, or 54 g of choline chloride. Treatments were preconditioned with ground corn and administered as a single ruminal bolus once per experimental period 1 h postfeeding of a total mixed ration. For experiment 2, we compared a control (0 g of choline chloride) versus P2, and P4 and P5 (60 and 62% choline chloride, respectively) in a repeated 4 × 4 Latin square design. Experiment 2 followed a similar design as experiment 1 with modifications: 12 late-lactation (228 ± 7.10 d in milk) Holstein cows were used; treatments were administered as part of a premeal; and cows received a daily allowance of a total mixed ration as equal provisions every 4 h within 24 h before and after treatment. For both experiments, plasma and milk samples were collected for choline and choline metabolite quantification. Data were analyzed using a mixed model including fixed effects of treatment, period, and time. Contrast statements were used to test for linearity of dose and differences between prototypes for experiment 1 and 2, respectively. Plasma and milk TMAO concentrations increased with RPC dose (peak by h). Milk choline and betaine yields increased with RPC dose in a quadratic manner; albeit, dependent upon RPC type. Milk phosphocholine (PCho) and glycerophosphorylcholine (GPC) yields changed by select RPC dose (experiment 1), however Met, PCho, GPC, phosphatidylcholine, and total choline concentrations in milk, and plasma Met and sphingomyelin concentrations were not responsive. We conclude that plasma or milk choline, betaine, and TMAO concentrations are responsive to RPC type, dose, and stage of lactation evaluated.
As part of the development of the beam imaging system at the European Spallation Source, luminescent screens have been fabricated by the flame spraying of scintillating materials onto stainless steel ...backings. A total of seven screens were produced, three of chromia alumina (Al2O3:Cr), two of YAG (Y3Al5O12:Ce) and two of a 50/50 mix of these. The properties of these screens under proton irradiation were evaluated using a 2.55MeV proton beam at currents of up to 10 μA. Irradiation times were up to 25 h per sample, during which luminescence-, spectrographic-, thermal- and current-data was sampled at a rate of 1Hz. Preliminary results of these measurements are reported here; with a quantitative analysis presented for one of the chromia alumina screens and a qualitative comparison of all three material types. The luminescent yield for chromia alumina was determined to be around 2000photons/MeV for a virgin screen, and was found to drop to 1.5% after 167mC of proton irradiation. A recovery of the luminescence of chromia alumina to >60% was observed after beam current was reduced for an 8 h period. Observations indicate that the YAG and mixed composition screens retain higher luminescence than the chromia alumina even at temperatures of over 200 °C. It is indicated that the luminescence from YAG feeds the R-lines of chromia alumina in the mixed composition screens.
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