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Abstract
Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single‐quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13C‐ and 32S‐labeled internal standards with external standard calibration confirmed the superiority of stable isotope‐labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2 = 0.945). In contrast to off‐line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2‐based extraction and separation techniques for potentially infective biomatrices.
Abstract
Neutrophils are the most abundant leukocytes in circulation playing a key role in acute inflammation during microbial infections. Phagocytosis, one of the crucial defence mechanisms of neutrophils against pathogens, is amplified by chemotactic leukotriene (LT)B4, which is biosynthesized via 5‐lipoxygenase (5‐LOX). However, extensive liberation of LTB4 can be destructive by over‐intensifying the inflammatory process. While enzymatic biosynthesis of LTB4 is well characterized, less is known about molecular mechanisms that activate 5‐LOX and lead to LTB4 formation during host–pathogen interactions. Here, we investigated the ability of the common opportunistic fungal pathogen Candida albicans to induce LTB4 formation in neutrophils, and elucidated pathogen‐mediated drivers and cellular processes that activate this pathway. We revealed that C. albicans‐induced LTB4 biosynthesis requires both the morphological transition from yeast cells to hyphae and the expression of hyphae‐associated genes, as exclusively viable hyphae or yeast‐locked mutant cells expressing hyphae‐associated genes stimulated 5‐LOX by [Ca2+]i mobilization and p38 MAPK activation. LTB4 biosynthesis was orchestrated by synergistic activation of dectin‐1 and Toll‐like receptor 2, and corresponding signaling via SYK and MYD88, respectively. Conclusively, we report hyphae‐specific induction of LTB4 biosynthesis in human neutrophils. This highlights an expanding role of neutrophils during inflammatory processes in the response to C. albicans infections.
Essential Oils as Multicomponent Mixtures and Their Potential for Human Health and Well-Being
(2022)
Essential oils (EOs) and their individual volatile organic constituents have been an inherent part of our civilization for thousands of years. They are widely used as fragrances in perfumes and cosmetics and contribute to a healthy diet, but also act as active ingredients of pharmaceutical products. Their antibacterial, antiviral, and anti-inflammatory properties have qualified EOs early on for both, the causal and symptomatic therapy of a number of diseases, but also for prevention. Obtained from natural, mostly plant materials, EOs constitute a typical example of a multicomponent mixture (more than one constituent substances, MOCS) with up to several hundreds of individual compounds, which in a sophisticated composition make up the property of a particular complete EO. The integrative use of EOs as MOCS will play a major role in human and veterinary medicine now and in the future and is already widely used in some cases, e.g., in aromatherapy for the treatment of psychosomatic complaints, for inhalation in the treatment of respiratory diseases, or topically administered to manage adverse skin diseases. The diversity of molecules with different functionalities exhibits a broad range of multiple physical and chemical properties, which are the base of their multi-target activity as opposed to single isolated compounds. Whether and how such a broad-spectrum effect is reflected in natural mixtures and which kind of pharmacological potential they provide will be considered in the context of ONE Health in more detail in this review.
Natural products comprise a rich reservoir for innovative drug leads and are a constant
source of bioactive compounds. To find pharmacological targets for new or already known
natural products using modern computer-aided methods is a current endeavor in drug discovery.
Nature’s treasures, however, could be used more effectively. Yet, reliable pipelines for the
large-scale target prediction of natural products are still rare. We developed an in silico workflow
Int. J. Mol. Sci. 2020, 21, 7102; doi:10.3390/ijms21197102 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2020, 21, 7102 2 of 18
consisting of four independent, stand-alone target prediction tools and evaluated its performance
on dihydrochalcones (DHCs)—a well-known class of natural products. Thereby, we revealed
four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1,
17β-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a
thorough strategy on how to perform computational target predictions and guidance on using the
respective tools.
Within the last decades cancer treatment improved by the availability of more specifically
acting drugs that address molecular target structures in cancer cells. However, those target-sensitive
drugs suffer from ongoing resistances resulting from mutations and moreover they are affected
by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be
treated with the common drugs, so that there have been long efforts to develop drugs to combat
that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in
cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective
efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those
inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4
we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a
MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition
of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active
compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant
anticancer drug as proof-of-principle to encourage for further preclinical studie
Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations
(2021)
Transcription factors play a crucial role in regulating biological processes such as cell
growth, differentiation, organ development and cellular signaling. Within this group, proteins
equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding
transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a
variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary
glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which
serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown
to be critically important for efficient regulation of the BCL11B target genes and could therefore
represent a promising target for novel drug therapies. Here, we report the structural basis for
BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure
prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy
transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of
the single ZF domain and directly involved in forming the dimer interface. These findings not only
provide deep insight into how BCL11B acquires its active structure but also represent an important
step towards rational design or selection of potential inhibitors.
Highly Virulent and Multidrug-Resistant Escherichia coli Sequence Type 58 from a Sausage in Germany
(2022)
Studies have previously described the occurrence of multidrug-resistant (MDR) Escherichia coli in human and veterinary medical settings, livestock, and, to a lesser extent, in the environment and food. While they mostly analyzed foodborne E. coli regarding phenotypic and sometimes genotypic antibiotic resistance and basic phylogenetic classification, we have limited understanding of the in vitro and in vivo virulence characteristics and global phylogenetic contexts of these bacteria. Here, we investigated in-depth an E. coli strain (PBIO3502) isolated from a pork sausage in Germany in 2021. Whole-genome sequence analysis revealed sequence type (ST)58, which has an internationally emerging high-risk clonal lineage. In addition to its MDR phenotype that mostly matched the genotype, PBIO3502 demonstrated pronounced virulence features, including in vitro biofilm formation, siderophore secretion, serum resilience, and in vivo mortality in Galleria mellonella larvae. Along with the genomic analysis indicating close phylogenetic relatedness of our strain with publicly available, clinically relevant representatives of the same ST, these results suggest the zoonotic and pathogenic character of PBIO3502 with the potential to cause infection in humans and animals. Additionally, our study highlights the necessity of the One Health approach while integrating human, animal, and environmental health, as well as the role of meat products and food chains in the putative transmission of MDR pathogens.
The study aimed to examine the influence of a rotating magnetic field (RMF) of two different frequencies (5 and 50 Hz) on the expression of regulatory (agrA, hld, rot) and staphylococcal enterotoxin (SE—sea, sec, sel) genes as well as the production of SEs (SEA, SEC, SEL) by the Staphylococcus aureus FRI913 strain cultured on a medium supplemented with a subinhibitory concentration of trans-anethole (TA). Furthermore, a theoretical model of interactions between the bacterial medium and bacterial cells exposed to RMF was proposed. Gene expression and SEs production were measured using quantitative real-time PCR and ELISA techniques, respectively. Based on the obtained results, it was found that there were no significant differences in the expression of regulatory and SE genes in bacteria simultaneously cultured on a medium supplemented with TA and exposed to RMF at the same time in comparison to the control (unexposed to TA and RMF). In contrast, when the bacteria were cultured on a medium supplemented with TA but were not exposed to RMF or when they were exposed to RMF of 50 Hz (but not to TA), a significant increase in agrA and sea transcripts as compared to the unexposed control was found. Moreover, the decreased level of sec transcripts in bacteria cultured without TA but exposed to RMF of 50 Hz was also revealed. In turn, a significant increase in SEA and decrease in SEC and SEL production was observed in bacteria cultured on a medium supplemented with TA and simultaneously exposed to RMFs. It can be concluded, that depending on SE and regulatory genes expression as well as production of SEs, the effect exerted by the RMF and TA may be positive (i.e., manifests as the increase in SEs and/or regulatory gene expression of SEs production) or negative (i.e., manifests as the reduction in both aforementioned features) or none.
Fibers and yarns are part of everyday life. So far, fibers that are also used pharmaceutically have mainly been produced by electrospinning. The common use of spinning oils and the excipients they contain, in connection with production by melt extrusion, poses a regulatory challenge for pharmaceutically usable fibers. In this publication, a newly developed small-scale direct-spinning melt extrusion system is described, and the pharmaceutically useful polyvinyl filaments produced with it are characterized. The major parts of the system were newly developed or extensively modified and manufactured cost-effectively within a short time using rapid prototyping (3D printing) from various materials. For example, a stainless-steel spinneret was developed in a splice design for a table-top melt extrusion system that can be used in the pharmaceutical industry. The direct processing of the extruded fibers was made possible by a spinning system developed called Spinning-Rosi, which operates continuously and directly in the extrusion process and eliminates the need for spinning oils. In order to prevent instabilities in the product, further modifications were also made to the process, such as a the moisture encapsulation of the melt extrusion line at certain points, which resulted in a bubble-free extrudate with high tensile strength, even in a melt extrusion line without built-in venting.