Synthesis and characterization of cytidine derivatives that inhibit the kinase IspE of the non-mevalonate pathway for isoprenoid biosynthesis

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co-crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co-crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well-conserved ATP-binding Gly-rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure-based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.     

Secreted production of a custom-designed, highly hydrophilic gelatin in Pichia pastoris

A custom-designed, highly hydrophilic gelatin was produced inPichia pastoris. Secreted production levels in single-copytransformants were in the range 3–6 g/l of clarified brothand purification to near homogeneity could be accomplished bydifferential ammonium sulfate precipitation. Despite the factthat gelatins are highly susceptible to proteolysis becauseof their unfolded structure, the recombinant protein was shownto be fully intact by SDS–PAGE, N-terminal sequencing,gel filtration chromatography and mass spectrometry. Owing toits highly hydrophilic nature, the migration of the syntheticgelatin in SDS–PAGE was severely delayed. Esterificationof the carboxylic amino acid side chains resulted in normalmigration. The high polarity of the synthetic gelatin also accountsfor its negligible surface activity in water at concentrationsup to 5% (w/v), as determined by tensiometry. Circular dichroismspectrometry showed that the non-hydroxylated gelatin did notform triple helices at 4°C. The spectrum was even more representativeof the random coil conformation than the spectrum of naturalnon-hydroxylated gelatins.     

Functionalizable composite nanoparticles as a dual magnetic resonance imaging/computed tomography contrast agent for medical imaging

A dual contrast agent for computed tomography (CT) and magnetic resonance imaging (MRI) was synthesized via microemulsion polymerization. This contrast agent consists of Fe₃O₄ particles (d = 7 nm) with an iodine-carrying nanopolymeric shell, with overall particle sizes ranging from 50 to 250 nm. 2-Methacryloyloxyethyl(2,3,5-triiodobenzoate) was used as the monomer. Sodium oleate was used as the surfactant and its amount was varied to control the overall particle size. The composite nanoparticles were mainly characterized via dynamic light scattering, with further analyses using transmission electron microscopy and atomic force microscopy. The particles provided a highly visible contrast in CT and MR images. A template for biomedical applications was created by adding a comonomer and the particles were further functionalized with the somatostatin analogue Tyr³-octreotate. The particles were tested for specific uptake into somatostatin receptor-positive AR42J cells. The additional uptake of the functionalized particles was investigated. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47571.     

Prozesstechnik auf der ACHEMA 2022

The ACHEMA 2022 was only half as large as usual, but the main trends in process technology were well represented. The research activities on the utilization of hydrogen and the use of CO₂, CO and polymer waste as raw materials were highlights. Furthermore, new developments in the design of heat exchangers, mixers and reactors, the connection of digitalization and process engineering, new solutions for energy efficiency as well as sophisticated measuring techniques and demonstrations of safety engineering made the ACHEMA an exciting fair.     

Toxic or beneficial? What is the role of food-relevant selenium species selenoneine?

Selenium (Se) is an essential trace element that is ubiquitously present in the environment in small concentrations. Essential functions of Se in the human body are manifested through the wide range of proteins, containing selenocysteine as their active center. Such proteins are called selenoproteins which are found in multiple physiological processes like antioxidative defense and the regulation of thyroid hormone functions. Therefore, Se deficiency is known to cause a broad spectrum of physiological impairments, especially in endemic regions with low Se content. Nevertheless, being an essential trace element, Se could exhibit toxic effects, if its intake exceeds tolerable levels. Accordingly, this range between deficiency and overexposure represents optimal Se supply. However, this range was found to be narrower than for any other essential trace element. Together with significantly varying Se concentrations in soil and the presence of specific bioaccumulation factors, this represents a noticeable difficulty in the assessment of Se epidemiological status. While Se is acting in the body through multiple selenoproteins, its intake occurs mainly in form of small organic or inorganic molecular mass species. Thus, Se exposure not only depends on daily intake but also on the respective chemical form, in which it is present. The essential functions of selenium have been known for a long time and its primary forms in different food sources have been described. Nevertheless, analytical capabilities for a comprehensive investigation of Se species and their derivatives have been introduced only in the last decades. A new Se compound was identified in 2010 in the blood and tissues of bluefin tuna. It was called selenoneine (SeN) since it is an isologue of naturally occurring antioxidant ergothioneine (ET), where Se replaces sulfur. In the following years, SeN was identified in a number of edible fish species and attracted attention as a new dietary Se source and potentially strong antioxidant. Studies in populations whose diet largely relies on fish revealed that SeN represents the main non-protein bound Se pool in their blood. First studies, conducted with enriched fish extracts, already demonstrated the high antioxidative potential of SeN and its possible function in the detoxification of methylmercury in fish. Cell culture studies demonstrated, that SeN can utilize the same transporter as ergothioneine, and SeN metabolite was found in human urine. Until recently, studies on SeN properties were severely limited due to the lack of ways to obtain the pure compound. As a predisposition to this work was firstly a successful approach to SeN synthesis in the University of Graz, utilizing genetically modified yeasts. In the current study, by use of HepG2 liver carcinoma cells, it was demonstrated, that SeN does not cause toxic effects up to 100 M concentration in hepatocytes. Uptake experiments showed that SeN is not bioavailable to the used liver cells. In the next part a blood-brain barrier (BBB) model, based on capillary endothelial cells from the porcine brain, was used to describe the possible transfer of SeN into the central nervous system (CNS). The assessment of toxicity markers in these endothelial cells and monitoring of barrier conditions during transfer experiments demonstrated the absence of toxic effects from SeN on the BBB endothelium up to 100 M concentration. Transfer data for SeN showed slow but substantial transfer. A statistically significant increase was observed after 48 hours following SeN incubation from the blood-facing side of the barrier. However, an increase in Se content was clearly visible already after 6 hours of incubation with 1 M of SeN. While the transfer rate of SeN after application of 0.1 M dose was very close to that for 1 M, incubation with 10 M of SeN resulted in a significantly decreased transfer rate. Double-sided application of SeN caused no side-specific transfer of SeN, thus suggesting a passive diffusion mechanism of SeN across the BBB. This data is in accordance with animal studies, where ET accumulation was observed in the rat brain, even though rat BBB does not have the primary ET transporter OCTN1. Investigation of capillary endothelial cell monolayers after incubation with SeN and reference selenium compounds showed no significant increase of intracellular selenium concentration. Speciesspecific Se measurements in medium samples from apical and basolateral compartments, as good as in cell lysates, showed no SeN metabolization. Therefore, it can be concluded that SeN may reach the brain without significant transformation. As the third part of this work, the assessment of SeN antioxidant properties was performed in Caco-2 human colorectal adenocarcinoma cells. Previous studies demonstrated that the intestinal epithelium is able to actively transport SeN from the intestinal lumen to the blood side and accumulate SeN. Further investigation within current work showed a much higher antioxidant potential of SeN compared to ET. The radical scavenging activity after incubation with SeN was close to the one observed for selenite and selenomethionine. However, the SeN effect on the viability of intestinal cells under oxidative conditions was close to the one caused by ET. To answer the question if SeN is able to be used as a dietary Se source and induce the activity of selenoproteins, the activity of glutathione peroxidase (GPx) and the secretion of selenoprotein P (SelenoP) were measured in Caco-2 cells, additionally. As expected, reference selenium compounds selenite and selenomethionine caused efficient induction of GPx activity. In contrast to those SeN had no effect on GPx activity. To examine the possibility of SeN being embedded into the selenoproteome, SelenoP was measured in a culture medium. Even though Caco-2 cells effectively take up SeN in quantities much higher than selenite or selenomethionine, no secretion of SelenoP was observed after SeN incubation. Summarizing, we can conclude that SeN can hardly serve as a Se source for selenoprotein synthesis. However, SeN exhibit strong antioxidative properties, which appear when sulfur in ET is exchanged by Se. Therefore, SeN is of particular interest for research not as part of Se metabolism, but important endemic dietary antioxidant.     

Peptidyl Vinyl Ketone Irreversible Inhibitors of Rhodesain: Modifications of the P2 Fragment

In this paper, we report the design, synthesis and biological investigation of a series of peptidyl vinyl ketones obtained by modifying the P2 fragment of previously reported highly potent inhibitors of rhodesain, the main cysteine protease of Trypanosoma brucei rhodesiense. Investigation of the structure–activity relationship led us to identify new rhodesain inhibitors endowed with an improved selectivity profile (a selectivity index of up to 22 000 towards the target enzyme), and/or an improved antitrypanosomal activity in the sub-micromolar range.     

Targeting Mitochondria in Diabetes

Type 2 diabetes (T2D), one of the most prevalent noncommunicable diseases, is often preceded by insulin resistance (IR), which underlies the inability of tissues to respond to insulin and leads to disturbed metabolic homeostasis. Mitochondria, as a central player in the cellular energy metabolism, are involved in the mechanisms of IR and T2D. Mitochondrial function is affected by insulin resistance in different tissues, among which skeletal muscle and liver have the highest impact on whole-body glucose homeostasis. This review focuses on human studies that assess mitochondrial function in liver, muscle and blood cells in the context of T2D. Furthermore, different interventions targeting mitochondria in IR and T2D are listed, with a selection of studies using respirometry as a measure of mitochondrial function, for better data comparison. Altogether, mitochondrial respiratory capacity appears to be a metabolic indicator since it decreases as the disease progresses but increases after lifestyle (exercise) and pharmacological interventions, together with the improvement in metabolic health. Finally, novel therapeutics developed to target mitochondria have potential for a more integrative therapeutic approach, treating both causative and secondary defects of diabetes.     

Influence of modern plastic furniture on the fire development in fires in homes: large‐scale fire tests in living rooms

About 80% of all fire fatalities in Germany occur because of fires in homes. It has been known for some time that modern materials (synonym for materials consisting mostly of synthetic polymers) tend to burn differently from older materials (synonym for materials consisting mostly of fibrous cellulosic substances) and it has been acknowledged that the amount of combustible plastics in homes has increased significantly over the last decades. To investigate the influence of modern furniture and ventilation conditions of fires in homes, a series of four large‐scale tests in two living rooms (LRs) with adjacent rooms (ARs) was performed by BAM and the Frankfurt fire service. Two LRs, one with older furniture and one with modern furniture, were tested twice each. Each test started with the ignition of a paper cushion on an upholstered chair. The influence of modern materials on the fire development was investigated, as well as the influence of the ventilation on the fire development. In all settings, an upholstered chair was the first burning item. Results of the test series show that fires in rooms with modern furniture develop faster than fires in rooms with older furniture. This is true for temperature development in the rooms as well as for smoke production.