Investigation of Bemethyl Biotransformation Pathways by Combination of LC–MS/HRMS and In Silico Methods

Bemethyl is an actoprotector, an antihypoxant, and a moderate psychostimulant. Even though the therapeutic effectiveness of bemethyl is well documented, there is a gap in knowledge regarding its metabolic products and their quantitative and qualitative characteristics. Since 2018, bemethyl is included to the Monitoring Program of the World Anti-Doping Agency, which highlights the challenge of identifying its urinary metabolites. The objective of the study was to investigate the biotransformation pathways of bemethyl using a combination of liquid chromatography-high-resolution mass spectrometry and in silico studies. Metabolites were analyzed in a 24 h rat urine collected after oral administration of bemethyl at a single dose of 330 mg/kg. The urine samples were prepared for analysis by a procedure developed in the present work and analyzed by high performance liquid chromatography–tandem mass spectrometry. For the first time, nine metabolites of bemethyl with six molecular formulas were identified in rat urine. The most abundant metabolite was a benzimidazole–acetylcysteine conjugate; this biotransformation pathway is associated with the detoxification of xenobiotics. The BioTransformer and GLORY computational tools were used to predict bemethyl metabolites in silico. The molecular docking of bemethyl and its derivatives to the binding site of glutathione S-transferase has revealed the mechanism of bemethyl conjugation with glutathione. The findings will help to understand the pharmacokinetics and pharmacodynamics of actoprotectors and to improve antihypoxant and adaptogenic therapy.     

Transamidation Down-Regulates Intestinal Immunity of Recombinant α-Gliadin in HLA-DQ8 Transgenic Mice

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120–139) and p23 (aa. 220–239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.     

Antibacterial agents and cystic fibrosis: synthesis and antimicrobial evaluation of a series of N-thiomethylazetidinones

The increasing emergence of multidrug-resistant microorganisms is one of the greatest challenges in the clinical management of infectious disease. New antimicrobial agents are therefore urgently required, particularly in the treatment of chronic and recurrent infections often associated with antibiotic-resistant pathogens, as in the case of cystic fibrosis (CF) patients. This study reports the antibacterial activity of a series of monocyclic β-lactams with an alkylidenecarboxyl chain or electron-withdrawing groups such as 4-OAc, 4-SAc, and 4-SO(2)Ph at the C4 position of the ring. N-Unsubstituted and N-thiomethyl derivatives were compared. A total of 33 azetidinones were tested for their activity against Gram-positive and Gram-negative bacterial clinical isolates. The combination of an N-thiomethyl group and a benzyl ester on the 4-alkylidene side chain were found to increase the potency against Gram-positive bacteria. The N-thiomethyl group clearly elevated the activity of 4-acetoxyazetidinones relative to the corresponding NH derivatives. The most active compounds showed minimum inhibitory concentration (MIC) values of 4 and 8 mg L(-1) against methicillin-resistant Staphylococcus aureus isolated from pediatric patients with CF.     

Mass spectrometry and the cellular surfaceome

The collection of exposed plasma membrane proteins, collectively termed the surfaceome, is involved in multiple vital cellular processes, such as the communication of cells with their surroundings and the regulation of transport across the lipid bilayer. The surfaceome also plays key roles in the immune system by recognizing and presenting antigens, with its possible malfunctioning linked to disease. Surface proteins have long been explored as potential cell markers, disease biomarkers, and therapeutic drug targets. Despite its importance, a detailed study of the surfaceome continues to pose major challenges for mass spectrometry-driven proteomics due to the inherent biophysical characteristics of surface proteins. Their inefficient extraction from hydrophobic membranes to an aqueous medium and their lower abundance compared to intracellular proteins hamper the analysis of surface proteins, which are therefore usually underrepresented in proteomic datasets. To tackle such problems, several innovative analytical methodologies have been developed. This review aims at providing an extensive overview of the different methods for surfaceome analysis, with respective considerations for downstream mass spectrometry-based proteomics.     

Fast capacitive probe for electromagnetic pulse diagnostic

In this work, we report the study and the development of a capacitive probe which is suitable for getting fast and high voltage/current measurements. Due to the fact that fast pulses propagate generally in coaxial structures, the probe realized in this work was a capacitive divider with the divider electrode properly designed to assure the same characteristic impedance of the coaxial structure and the recombination time of the split signals during the propagation. It was a folded cylindrical ring of 1.4 cm long and 0.8 cm thick, which introduce a theoretical delay time of about 100 ps. Analyzing the behavior of the probe closed on 520 Omega, the voltage amplification resulted to be of (3.6+/-0.1) x 10(-4) and, as a consequence, the current attenuation factor of 56+/-1 AV. The response rise time was less than 320 ps, which was limited by oscilloscope bandwave. The capacitor probe can operate voltage measurements of the order of 100 kV.     

Balancing–sequencing procedure for a mixed model assembly system in case of finite buffer capacity

In the last decades, the necessity to make production more versatile and flexible has forced assembly line production systems to change from fixed assembly lines to mixed model assembly lines, where the output products are variations of the same base product and only differ in specific customizable attributes. Such assembly lines allow reduced setup time, since products can be jointly manufactured in intermixed sequences (Boysen, Flieder, Scholl. Jena Research Papers in Business and Economics, Friedrich-Schiller-Universitat Jena, 1;1–11, 2007a; Boysen, Flieder, Scholl. Jena Research Papers in Business and Economics, Friedrich-Schiller-Universitat Jena, 2;1–33, 2007b). Unfortunately, the installation of customization options typically leads to variations in process times, and when the cycle is exceeded within a certain station, an overload is created, forcing other stations to wait and idle. Normally, process time variation in an un-paced line are absorbed by buffers, but in some industrial application the buffer dimensions are critical not only for the reduction of work in progress but also in reducing other constrains (space, technology, model dimensions, etc.). The problem of balancing mixed model assembly lines (MALBP), in the long term, and that of sequencing mixed model assembly lines (MMS), in the short term (Merengo, Nava, Pozetti. Int J Prod Res 37:2835–2860, 1999), are the two major problems to solve. The object of this paper is to illustrate an innovative balancing–sequencing step-by-step procedure that aims to optimize the assembly line performance and at the same time contain the buffer dimensions in function of different market demand and production mix. The model is validated using a simulation software and an industrial application is presented.     

Order picking system design: the storage assignment and travel distance estimation (SA&TDE) joint method

Of all the warehouse activities, order picking is one of the most time-consuming and expensive. In order to improve the task, several researches have pointed out the need to consider jointly the layout of the warehouse, the storage assignment strategy and the routing policy to reduce travelled distances and picking time. This paper presents the storage assignment and travel distance estimation (SA&TDE) joint method, a new approach useful to design and evaluate a manual picker-to-parts picking system, focusing on goods allocation and distances estimation. Starting from a set of picking orders received in a certain time range, this approach allows to evaluate the combinations of product codes assigned to storage locations, aisles, sections or warehouse areas and to assess the most relevant ones, for the best location and warehouse layout, with the aim of ensuring optimal picking routes, through the application of the multinomial probability distribution. A case study is developed as well, in order to clarify the concept that underlies the SA&TDE joint method, and to show the validity and the flexibility of the approach, through the calculation of the saving at different levels of detail.