Top-down characterization of nucleic acids modified by structural probes using high-resolution tandem mass spectrometry and automated data interpretation

A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated reduction and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data reduction module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calculates the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data reduction program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on cross-linking strategies and top-down ESI-FTMS.     

FTMS structure elucidation of natural products: application to muraymycin antibiotics using ESI multi-CHEF SORI-CID FTMS(n), the top-down/bottom-up approach,and HPLC ESI capillary-skimmer CID FTMS

The molecular formulas for the structures and substructures of muraymycin antibiotics A1 (C52H90N14O19, MW 1214) and B1 (C49H83N11O18, MW 1113) were determined using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The muraymycin A1 and B1 structures were elucidated by utilizing capillary-skimmer fragmentation with up to five stages of mass spectrometry (MS5). Multi-CHEF, a multiple ion isolation method, was used at each stage of MS(n) to isolate a parent ion and up to four reference ions, for exact-mass calibration. The parent ions were fragmented by SORI-CID and the product ions internally calibrated with average absolute mass errors less than 1 ppm at each stage in the fragmentation processes. Using the top-down/bottom-up approach, the molecular formulas for the antibiotics were determined by summing the elemental formulas of the neutral losses, obtained by measuring the mass differences (<500 Da) between the genetically related sequential parent ion masses in the MS(n) spectra, with the unique elemental formula of the lowest parent ion mass (<500 Da). The structures of 12 additional compounds in the muraymycin complex were elucidated using HPLC ESI capillary-skimmer CID FTMS by correlating their fragmentation patterns with those of muraymycins A1 and B1. Sequential neutral losses of an aminosugar, a valine, a uridine, and an ester fatty acid from the muraymycin parent ions provided diagnostic fragments for characterization.     

Consumer credit risk: Individual probability estimates using machine learning

Consumer credit scoring is often considered a classification task where clients receive either a good or a bad credit status. Default probabilities provide more detailed information about the creditworthiness of consumers, and they are usually estimated by logistic regression. Here, we present a general framework for estimating individual consumer credit risks by use of machine learning methods. Since a probability is an expected value, all nonparametric regression approaches which are consistent for the mean are consistent for the probability estimation problem. Among others, random forests (RF), k-nearest neighbors (kNN), and bagged k-nearest neighbors (bNN) belong to this class of consistent nonparametric regression approaches. We apply the machine learning methods and an optimized logistic regression to a large dataset of complete payment histories of short-termed installment credits. We demonstrate probability estimation in Random Jungle, an RF package written in C++ with a generalized framework for fast tree growing, probability estimation, and classification. We also describe an algorithm for tuning the terminal node size for probability estimation. We demonstrate that regression RF outperforms the optimized logistic regression model, kNN, and bNN on the test data of the short-term installment credits.     

Assessment of cytotoxicity and immune compatibility of phytochemicals‐mediated biosynthesised silver nanoparticles using Cynara scolymus

The study was focused on the phytochemicals‐mediated biosynthesis of silver nanoparticles using leaf extracts and infusions from Cynara scolymus. To identify the antioxidant activity and total phenolic content, the 1,1‐diphenyl‐1‐picrylhydrazyl and Folin–Ciocalteau methods were applied, respectively. The formation and stability of the reduced silver ions were monitored by UV–vis spectrophotometer. The particle sizes of the silver nanoparticles were characterised using the dynamic light scattering technique and scanning electron microscope. The phase composition of the obtained silver nanoparticles was characterised by X‐ray diffraction. The silver nanoparticles suspension, artichoke infusion, and silver ions were separately tested towards potential cytotoxicity and pro‐inflammatory effect using mouse fibroblasts and human monocytes cell line, respectively. The total phenolic content and antioxidant activity of ethanol extract and infusion were found significantly higher as compared to aqueous extract and infusion. The UV–visible spectrophotometric analysis revealed the presence of the characteristic absorption band of the Ag nanoparticles. Moreover, it was found that with the increasing volume of plant extract, the average size of particles was increased. Biocompatibility results evidently showed that silver nanoparticles do not induce monocyte activation, however in order to avoid their cytotoxicity suspension at a concentration <2 ppm should be applied.Inspec keywords: pharmaceuticals, health and safety, renewable materials, toxicology, organic compounds, antibacterial activity, X‐ray diffraction, nanomedicine, nanoparticles, nanofabrication, suspensions, ultraviolet spectra, visible spectra, scanning electron microscopy, silver, particle sizeOther keywords: phytochemicals‐mediated biosynthesis, antioxidant activity, total phenolic content, dynamic light scattering technique, silver nanoparticles suspension, scanning electron microscopy, Cynara scolymus, 1,1 diphenyl‐1‐picrylhydrazyl method, cytotoxicity, immune compatibility, leaf extracts, UV‐vis spectrophotometry, particle size, Folin‐Ciocalteau methods, phase composition, X‐ray diffraction, artichoke infusion, pro‐inflammatory effect, mouse fibroblasts, human monocytes cell line, Ag     

The Potential of Asiatic Acid in the Reversion of Cyclophosphamide-Induced Hemorrhagic Cystitis in Rats

The purpose of this study was to determine if asiatic acid may act efficiently in the model of cyclophosphamide (CYP)-induced cystitis in rats. We performed experiments after administration of CYP (single dose 200 mg/kg, intraperitoneally), asiatic acid (30 mg/kg/day for 14 consecutive days, by oral gavage), or CYP plus asiatic acid, during which conscious cystometry, measurements of urothelium thickness and bladder edema, as well as selected biomarkers analyses were conducted. In rats that received asiatic acid together with CYP, a drop in bladder basal pressure, detrusor overactivity index, non-voiding contraction amplitude, non-voiding contraction frequency, and the area under the pressure curve were observed, when compared to the CYP group. Furthermore, a significant increase in threshold pressure, voided volume, intercontraction interval, bladder compliance, and volume threshold to elicit NVC were found in that group accordingly. Administration of the asiatic acid successfully restored concentrations of biomarkers both in bladder urothelium (BDNF, CGRP, OCT-3, IL-1β, IL-6, NGF, nitrotyrosine, malondialdehyde, TNF-α, SV2A, SNAP23, SNAP25, PAC-1, ORM1, occludin, IGFBP-3, HB-EGF, T–H protein, Z01, and HPX) and detrusor muscle (Rho kinase and VAChT) in CYP-treated rats. Finally, asiatic acid significantly decreased urothelium thickness and bladder oedema. Asiatic acid proved to be a potent and effective drug in the rat model of CYP-induced cystitis.     

Ion implantation of Si in semi-insulating In0.53Ga0.47As:Fe

Ion implantation of Si in semi-insulating In0.53Ga0.47As:Fe was discussed. Electrical activation of more than 100%, a broad implant profile and an average electron mobility of 3000 cm2/Vs are observed in layers implanted with a dose of 2 × 1013 cm?2 at 260 keV and annealed at 670°C for 15 min. The results of photoluminescence measurements are also presented.     

Highly Stretchable Conducting SIBS‐P3HT Fibers

Poly(styrene‐β‐isobutylene‐β‐styrene)‐poly(3‐hexylthiophene) (SIBS‐P3HT) conducting composite fibers are successfully produced using a continuous flow approach. Composite fibers are stiffer than SIBS fibers and able to withstand strains of up 975% before breaking. These composite fibers exhibit interesting reversible mechanical and electrical characteristics, which are applied to demonstrate their strain gauging capabilities. This will facilitate their potential applications in strain sensing or elastic electrodes. Here, the fabrication and characterization of highly stretchable electrically conducting SIBS‐P3HT fibers using a solvent/non‐solvent wet‐spinning technique is reported. This fabrication method combines the processability of conducting SIBS‐P3HT blends with wet‐spinning, resulting in fibers that could be easily spun up to several meters long. The resulting composite fiber materials exhibit an increased stiffness (higher Young’s modulus) but lower ductility compared to SIBS fibers. The fibers’ reversible mechanical and electrical characteristics are applied to demonstrate their strain gauging capabilities.