Rugulactone and its analogues exert antibacterial effects through multiple mechanisms including inhibition of thiamine biosynthesis

Rugulactone is a dihydro-α-pyrone isolated from the plant Cryptocarya rugulosa in 2009. It has been reported to display IkB kinase (IKK) inhibitory activity, as well as antibiotic activity in several strains of pathogenic bacteria. However, its biological targets and mode of action in bacteria have not yet been explored. Here we present enantioselective syntheses of rugulactone and of some corresponding activity-based protein profiling (ABPP) probes. We found that the ABPP probes in this study are more potent than rugulactone against Staphyloccocus aureus NCTC 8325, S. aureus Mu50, Listeria welshimeri SLCC 5334 and Listeria monocytogenes EGD-e, and that molecules of this class probably exert their antibacterial effect through a combination of targets. These targets include covalent inhibition of 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMPP) kinase (ThiD), which is an essential component of the thiamine biosynthesis pathway in bacteria. This represents the first example of a small-molecule inhibitor of ThiD.     

Imaging of Extracellular pH Using Hyperpolarized Molecules

Many diseases can overrule natural pH regulatory mechanisms and alter the extracellular pH (pH_e). A non-invasive method that resolves pH_e in vivo with high spatial and temporal resolution could therefore improve diagnosis and monitoring of diseases, contributing to the concept of precision medicine. During the last decades, several techniques have been proposed to image pH_e non-invasively. The majority of these methods rely on magnetic resonance because of its good spatial resolution, high penetration depth, non-ionizing radiation and excellent complimentary soft tissue contrast. Dissolution dynamic nuclear polarization (DNP) is an emerging concept to enhance nuclear magnetic resonance (NMR) signals by more than four orders of magnitude, making it possible to observe in vivo metabolic processes in real-time. Here, we summarize and review recent developments in pH_e imaging techniques based on hyperpolarization methods and give an overview of recently discovered hyperpolarized pH sensor molecules that have been applied in vitro and in vivo.     

Advances in Transgenic Mouse Models to Study Infections by Human Pathogenic Viruses

Medical research is changing into direction of precision therapy, thus, sophisticated preclinical models are urgently needed. In human pathogenic virus research, the major technical hurdle is not only to translate discoveries from animals to treatments of humans, but also to overcome the problem of interspecies differences with regard to productive infections and comparable disease development. Transgenic mice provide a basis for research of disease pathogenesis after infection with human-specific viruses. Today, humanized mice can be found at the very heart of this forefront of medical research allowing for recapitulation of disease pathogenesis and drug mechanisms in humans. This review discusses progress in the development and use of transgenic mice for the study of virus-induced human diseases towards identification of new drug innovations to treat and control human pathogenic infectious diseases.     

7-(Dialkylamino)coumarin-4-yl]methyl-Caged Compounds as Ultrafast and Effective Long-Wavelength Phototriggers of 8-Bromo-Substituted Cyclic Nucleotides

[7-(Dimethylamino)coumarin-4-yl]methyl (DMACM) and [7-(diethylamino)coumarin-4-yl]methyl (DEACM) esters of 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) and 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) are described as novel caged compounds for 8-bromo-substituted cyclic nucleotides. Synthesis is accomplished by treatment of the free acids of the cyclic nucleotides with the corresponding 7(dialkylamino)-substituted 4(diazomethyl)coumarins. Irradiation of the DMACM- and DEACM-caged cyclic nucleotides with UV light stimulates the release of the cyclic nucleotides within roughly a nanosecond. The new caged compounds are resistant to hydrolysis in aqueous buffers and exhibit long-wavelength absorption properties with maxima at 400 nm, high extinction coefficients, and high quantum yields (0.15-0.31). Their favorable properties render these compounds the most efficient and rapid phototriggers of 8-bromo-substituted cyclic nucleotides known. The usefulness of the compounds for physiological studies under nondamaging light conditions was examined in HEK293 cells expressing the alpha subunit of the cyclic-nucleotide-gated (CNG) channel of cone photoreceptors (CNGA3) and of olfactory neurons (CNGA2) by using confocal laser scanning microscopy and the patch clamp technique.     

Adventitial Tertiary Lymphoid Organs as Potential Source of MicroRNA Biomarkers for Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is an inflammatory disease associated with marked changes in the cellular composition of the aortic wall. This study aims to identify microRNA (miRNA) expression in aneurysmal inflammatory cells isolated by laser microdissection from human tissue samples. The distribution of inflammatory cells (neutrophils, B and T lymphocytes, mast cells) was evaluated in human AAA biopsies. We observed in half of the samples that adventitial tertiary lymphoid organs (ATLOs) with a thickness from 0.5 to 2 mm were located exclusively in the adventitia. Out of the 850 miRNA that were screened by microarray in isolated ATLOs (n = 2), 164 miRNAs were detected in ATLOs. The three miRNAs (miR-15a-3p, miR-30a-5p and miR-489-3p) with the highest expression levels were chosen and their expression quantified by RT-PCR in isolated ATLOs (n = 4), M1 (n = 2) and M2 macrophages (n = 2) and entire aneurysmal biopsies (n = 3). Except for the miR-30a-5p, a similar modulation was found in ATLOs and the two subtypes of macrophages. The modulated miRNAs were then evaluated in the plasma of AAA patients for their potential as AAA biomarkers. Our data emphasize the potential of miR-15a-3p and miR-30a-5p as biomarkers of AAA but also as triggers of ATLO evolution. Further investigations will be required to evaluate their targets in order to better understand AAA pathophysiology.     

Different Enzymatic Processing of γ‐Phosphoramidate and γ‐Phosphoester‐Modified ATP Analogues

Monitoring the activity of ATP‐consuming enzymes provides the basis for elucidating their modes of action and regulation. Although a number of ATP analogues have been developed for this, their scope is restricted because of the limited acceptance by respective enzymes. In order to clarify which kind of phosphate‐modified ATP analogues are accepted by the α‐β‐phosphoanhydride‐cleaving ubiquitin‐activating enzyme 1 (UBA1) and the β‐γ‐phosphoanhydride‐cleaving focal adhesion kinase (FAK), we tested phosphoramidate‐ and phosphoester‐modified ATP analogues. UBA1 and FAK were able to convert phosphoramidate‐modified ATP analogues, even with a bulky modification like biotin. In contrast, a phosphoester‐modified analogue was poorly accepted. These results demonstrate that minor variations in the design of ATP analogues for monitoring ATP utilization have a significant impact on enzymatic acceptance.     

Controlling Cellular Uptake and Toxicity of Polyphenylene Dendrimers by Chemical Functionalization

Polyphenylene dendrimers (PPDs) represent a unique class of macromolecules based on their monodisperse and shape‐persistent nature. These characteristics have enabled the synthesis of a new genre of “patched” surface dendrimers, where their exterior can be functionalized with a variety of polar and nonpolar substituents to yield lipophilic binding sites in a site‐specific way. Although such materials are capable of complexing biologically relevant molecules, show high cellular uptake in various cell lines, and low to no toxicity, there is minimal understanding of the driving forces to these characteristics. We investigated whether it is the specific chemical functionalities, relative quantities of each moiety, or the “patched” surface patterning on the dendrimers that more significantly influences their behavior in biological media.     

Small‐Molecule Inhibitors of the Tumor Suppressor Fhit

The tumor suppressor Fhit and its substrate diadenosine triphosphate (Ap₃A) are important factors in cancer development and progression. Fhit has Ap₃A hydrolase activity and cleaves Ap₃A into adenosine monophosphate (AMP) and adenosine diphosphate (ADP); this is believed to terminate Fhit‐mediated signaling. How the catalytic activity of Fhit is regulated and how the Fhit ? Ap₃A complex might exert its growth‐suppressive function remain to be discovered. Small‐molecule inhibitors of the enzymatic activity of Fhit would provide valuable tools for the elucidation of its tumor‐suppressive functions. Here we describe the development of a high‐throughput screen for the identification of such small‐molecule inhibitors of Fhit. Two clusters of inhibitors that decreased the activity of Fhit by at least 90 % were identified. Several derivatives were synthesized and exhibited in vitro IC₅₀ values in the nanomolar range.     

Performance Enhancement of Polycondensate‐Based Superplasticizers by Encapsulation

A cost‐effective and robust encapsulation system for chemical admixtures with delayed release mechanisms for construction materials like dry mix mortars is presented. Based on supplementary cementitious materials, a superplasticizer was encapsulated in matrix‐based encapsulations. Subsequently, the particle characteristics of the agglomerates and the release behavior of the superplasticizer were examined. The main objective of this research was to prove the functionality of such encapsulations for the future use in construction materials. The results obtained indicate that the performance of construction chemicals could be improved by encapsulation and controlled release for the future development of new construction materials. Furthermore, material characteristics of the supplementary cementitious material like the incline to excessive dusting or the materials density were improved by encapsulation.