Double Strain‐Promoted Macrocyclization for the Rapid Selection of Cell‐Active Stapled Peptides

Peptide stapling is a method for designing macrocyclic alpha‐helical inhibitors of protein–protein interactions. However, obtaining a cell‐active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain‐promoted azide–alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell‐active stapled peptides. As a proof of concept, MDM2‐binding peptides were stapled in parallel, directly in cell culture medium in 96‐well plates, and simultaneously evaluated in a p53 reporter assay. This in situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α‐Helicity was confirmed by a crystal structure of the MDM2‐peptide complex. This work introduces in situ stapling as a versatile biocompatible technique with many other potential high‐throughput biological applications.     

In vitro antioxidant and inhibitory activity of water decoctions of carob tree (Ceratonia siliqua L.) on cholinesterases, α-amylase and α-glucosidase

This work reports the in vitro inhibitory activity of water decoctions of leaves, germ flour, pulp, locust bean gum and stem bark of carob tree on α-amylase, α-glucosidase, acetylcholinesterase and butyrylcholinesterase. The antioxidant activity and the chemical characterisation of the extracts made by spectrophotometric assays and by high-performance liquid chromatography are also reported. Leaves and stem bark decoctions strongly inhibited all the enzymes tested, had significant antioxidant activity and the highest total phenolics content. The major compounds were identified as gallic acid in the leaves and gentisic acid in the stem bark.     

Nature of the antimicrobial activity of Lactobacillus caseiBifidobacterium bifidum and Bifidobacterium animalis against foodborne pathogenic and spoilage microorganisms

The antimicrobial activities as well as the nature of the inhibitory compounds obtained from Lactobacillus casei, Bifidobacterium bifidum and Bifidobacterium animalis strains were assayed on foodborne pathogenic – Staphyloccoccus aureus subsp. aureus (CCUG ATCC® 25926?) and Escherichia coli (ATCC® 25922?) – and spoilage microorganisms – Pseudomonas aeruginosa (ATCC® 27853?). Test producer strains showed inhibitory effect on all indicator microorganisms in diffusion of cell-free concentrated supernatant by agar in well methods (10.0–22.5 mm) in periods of 24, 48 and 72 h. Inhibitory compounds showed no sensitivity to the action of proteolytic enzyme trypsin and were completely inactivated by adjusting the pH of the cell-free 20 × concentrated supernatant to 7.0. The results demonstrated that antimicrobial substances do not have proteinaceous nature and are caused by the action of organic acids with decreasing medium pH.     

Quantification of nitrogen compounds in diesel fuel samples by comprehensive two‐dimensional gas chromatography coupled with quadrupole mass spectrometry

Although several methods for the analysis of nitrogen compounds in diesel fuel have been described in the literature, the demand for rapid, sensitive, and robust analyses has increased in recent years. In this study, a comprehensive two‐dimensional gas chromatographic method was developed for the identification and quantification of nitrogen compounds in diesel fuel samples. The quantification was performed using the standard addition method and the analysis was conducted using comprehensive two‐dimensional gas chromatography coupled with fast quadrupole mass spectrometry. This study is the first to report quantification of nitrogen compounds in diesel fuel samples using the standard addition method without fractionation. This type of analysis was previously performed using many laborious separation steps, which can lead to errors and losses. The proposed method shows good linearity for target nitrogen compounds evaluated (m‐toluidine, 4‐ethylaniline, indole, 7‐methylindole, 7‐ethylindole, carbazole, isoquinoline, 4‐methylquinoline, benzo[h]quinolone, and acridine) over a range from 0.05 to 2.0 mg/L, and limits of detection and quantification of <0.06 and 0.16 mg/L, respectively, for all nitrogen compounds studied.     

Effects of Neutralization Process on Preparation and Characterization of Chitosan Membranes for Wound Dressing

Summary: Neutralization process effects on preparation and characterization of chitosan membranes were evaluated by differencial scanning calorimetry and scanning electron microscopy. Water solubility and humidity of chitosan membranes were also studied. Swelling behavior in different pH media was evaluated and mechanical properties such as tensile strength and elongation at break were measured. Neutralization process increased glass transition temperature of chitosan membranes and decreased their water solubility, humidity and water sorption. An improvement in mechanical properties of chitosan membranes was also observed after neutralization process.     

Detection of prior irradiation of dried fruits by electron spin resonance (ESR)

Dried almonds, raisins, dates and pistachio were irradiated using either gamma radiation or electron beam, at an average absorbed dose of 5 kGy. To detect the previous irradiation different parts of the dried fruits were analyzed by ESR spectroscopy: almonds: skin; raisins: dried pulp; dates: dried pulp and stone; pistachio: nutshell. Analyses were carried out 2–3 months and 6 months after irradiation. A series of signals tentatively described as “cellulose-like”, “sugar-like” and “complex” radical were observed, and some slight differences between spectra from samples irradiated with gamma rays and electrons were evident.     

Local field correction PIV: on the increase of accuracy of digital PIV systems

Cross-correlation Particle Image Velocimetry (PIV) has become a well known and widely used experimental technique. It has been already documented that difficulties arise resolving velocity structures smaller than the interrogation window. This is caused by signal averaging over this window. A new cross-correlation PIV method that eliminates this restriction is presented. The new method brings some other enhancements, such as the ability to deal with large velocity gradients, seeding density inhomogeneities, and high dispersion in the brightness of the particles. The final result is a method with a remarkable capability for accurately resolving small scale structures in the flow, down to a few times the mean distance between particles. When compared to particle tracking velocimetry, the new method is capable of obtaining measurements at high seeding density concentrations. Therefore, better overall performance is obtained, especially with the limited resolutions of video CCDs. In this paper, the new method is described and its performance is evaluated and compared to traditional PIV systems using synthetic images. Application to real PIV data are included and the results discussed. Received: 9 March 1998 / Accepted: 25 August 1998