Development of Tyrphostin Analogues to Study Inhibition of the Mycobacterium tuberculosis Pup Proteasome System**

Tuberculosis is a global health problem caused by infection with the Mycobacterium tuberculosis (Mtb) bacteria. Although antibiotic treatment has dramatically reduced the impact of tuberculosis on the population, the existence and spreading of drug resistant strains urgently demands the development of new drugs that target Mtb in a different manner than currently used antibiotics. The prokaryotic ubiquitin-like protein (Pup) proteasome system is an attractive target for new drug development as it is unique to Mtb and related bacterial genera. Using a Pup-based fluorogenic substrate, we screened for inhibitors of Dop, the Mtb depupylating protease, and identified I-OMe-Tyrphostin AG538 ( 1 ) and Tyrphostin AG538 ( 2 ). The hits were validated and determined to be fast-reversible, non-ATP competitive inhibitors. We synthesized >25 analogs of 1 and 2 and show that several of the synthesized compounds also inhibit the depupylation actions of Dop on native substrate, FabD-Pup. Importantly, the pupylation activity of PafA, the sole Pup ligase in Mtb, was also inhibited by some of these compounds.     

Revisiting Steroidogenic Pathways in the Human Placenta and Primary Human Trophoblast Cells

Steroid hormones play a crucial role in supporting a successful pregnancy and ensuring proper fetal development. The placenta is one of the principal tissues in steroid production and metabolism, expressing a vast range of steroidogenic enzymes. Nevertheless, a comprehensive characterization of steroidogenic pathways in the human placenta and potential developmental changes occurring during gestation are poorly understood. Furthermore, the specific contribution of trophoblast cells in steroid release is largely unknown. Thus, this study aimed to (i) identify gestational age-dependent changes in the gene expression of key steroidogenic enzymes and (ii) explore the role of trophoblast cells in steroid biosynthesis and metabolism. Quantitative and Droplet Digital PCR analysis of 12 selected enzymes was carried out in the first trimester (n = 13) and term (n = 20) human placentas. Primary trophoblast cells (n = 5) isolated from human term placentas and choriocarcinoma-derived cell lines (BeWo, BeWo b30 clone, and JEG-3) were further screened for gene expression of enzymes involved in placental synthesis/metabolism of steroids. Finally, de novo steroid synthesis by primary human trophoblasts was evaluated, highlighting the functional activity of steroidogenic enzymes in these cells. Collectively, we provide insights into the expression patterns of steroidogenic enzymes as a function of gestational age and delineate the cellular origin of steroidogenesis in the human placenta.     

Extraction of polyphenolic substances from bark as natural colorants for wool dyeing

In Europe, considerable amounts of bark are available from wood‐processing industries such as forestry and timber production. Polyphenolic components can be collected by hot water extraction. The extracted compounds can then be applied as colorants in textile dyeing operations. In this study, a comparative assessment of four different tree species with regard to their colouristic potential for wool dyeing was performed. Aqueous extracts from alder, ash tree, spruce and oak bark were prepared and analysed for their total phenolic content and ultraviolet (UV) absorption at 360–370 nm. The extracts were used for meta‐mordant dyeing by adding iron sulphate mordant (FeSO₄ × 7H₂O). For comparison, iron salt‐based dye lakes were prepared and used in dyeing experiments. For each tree species, a specific correlation between the total phenolic content of the dyebath and the colour depth in terms of K/S and CIELab coordinates was observed, both for the aqueous extracts and the dye lakes. Based on this relationship, standardisation and quality control of raw materials and dye lakes can be installed as important stages in the industrialisation of natural colorants from bark. The preparation of concentrated dye lakes permits formation of a concentrated colorant as dye product, which then can be standardised and delivered to textile dyehouses, similar to synthetic dyes. The preparation of dye lakes offers a relevant route towards achieving the commercialisation of bark extracts as natural colorants.     

High-throughput,high-sensitivity genetic mutation detection by tandem single-strand conformation polymorphism/heteroduplex analysis capillary array electrophoresis

We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.     

Rapid differentiation of new apple cultivars by headspace solid-phase microextraction in combination with chemometrical data processing

The aim of this study was to test a combination of automated headspace solid phase-microextraction gas chromatography (GC) with chemometrical data treatment for the rapid differentiation of enzyme-inactivated homogenates of new apple cultivars. The four cultivars Pinova, Piflora, Renora and Florina are characterized by different volatile patterns. Differences in the contents of volatiles were especially found for butyl acetate, ethyl butanoate, 2-methyl butanol, ethyl acetate and 6-methyl-5-hepten-2-ol. The used sample preparation method for GC coupled with pattern recognition of chromatograms is a useful tool for rapid and reliable determination of large numbers of samples.     

Implementation of a telemonitoring system for the control of an EEG-based brain-computer interface

By the use of a brain-computer interface (BCI), it is possible for completely paralyzed patients, who have lost their ability to speak, to have a new possibility to communicate with their environment. The training with such a BCI system can be performed at the patient's home, if there is a responsible person present who is familiar with the system. This person has to adjust different parameters and to adapt the training individually to each patient. Since this function is usually taken over by the developers of the system, the number of patients who can be included in regular BCI training is restricted due to geographical distances. This paper describes the implementation of a telemonitoring system, which makes it possible for the developer to control and supervise the BCI training from his or her own place of work. First experiences with a patient living far away from the developer's lab are reported.