Optimization of oncocin for antibacterial activity using a SPOT synthesis approach: extending the pathogen spectrum to Staphylococcus aureus.

The identification of lead molecules against multidrug-resistant bacteria ensuing the development of novel antimicrobial drugs is an urgent task. Proline-rich antimicrobial peptides are highly active in vitro and in vivo, but only against a few Gram-negative human pathogens, with rather weak activities against Pseudomonas aeruginosa and Staphylococcus aureus. This reduced level of efficacy could be related to inadequate uptake mechanisms or structural differences of the intracellular target proteins, i.e., the 70S ribosome or chaperone DnaK. Here we synthesized peptide arrays on cellulose membranes using cleavable linkers to release the free individual peptides for further antimicrobial tests. Thus, a library of singly substituted oncocin analogs was produced by replacing each residue by all other 19 canonical amino acids yielding a set of 361 individual peptides to be evaluated against a luminescent P. aeruginosa strain. Thirteen substitutions appeared promising and their improved antibacterial activities were confirmed for different bacteria after larger scale synthesis of these analogs. By combining two favorable substitutions into one peptide, we finally obtained an oncocin analog that was ten times more active against P. aeruginosa and even 100-fold more active against S. aureus than the original oncocin, providing minimal inhibitory concentrations of 4-8 and 0.5 µg/mL, respectively.

Decoration of silk fibroin by click chemistry for biomedical application.

Silkfibroin (SF) has an excellent biocompatibility and its remarkable structure translates into exciting mechanical properties rendering this biomaterial particularly fascinating for biomedical application. To further boost the material's biological/preclinical impact, SF is decorated with biologics, typically by carbodiimide/N-hydroxysuccinimide coupling (EDC/NHS). For biomedical application, this chemistry challenges the product risk profile due to the formation of covalent aggregates, particularly when decoration is with biologics occurring naturally in humans as these aggregates may prime for autoimmunity. Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry) provides the necessary specificity to avoid such intermolecular, covalent aggregates. We present a blueprint outlining the necessary chemistry rendering SF compatible with CuAAC and with a particular focus on structural consequences. For that, the number of SF carboxyl groups (carboxyl-SF; required for EDC/NHS chemistry) or azido groups (azido-SF; required for click chemistry) was tailored by means of diazonium coupling of the SF tyrosine residues. Structural impact on SF and decorated SF was characterized by Fourier transform infrared spectroscopy (FTIR). The click chemistry yielded a better controlled product as compared to the EDC/NHS chemistry with no formation of inter- and intramolecular crosslinks as demonstrated for SF decorated with fluorescent model compounds or a biologic, fibroblast growth factor 2 (FGF2), respectively. In conclusion, SF can readily be translated into a scaffold compatible with click chemistry yielding decorated products with a better risk profile for biomedical application.

Experimental measles encephalitis in Lewis rats: dissemination of infected neuronal cell subtypes.

Acute measles may lead in rare instances to the chronic progressive central nervous system disease process subacute sclerosing panencephalitis (SSPE). SSPE results from a persistent measles virus (MV) infection with incomplete virus replication involving the entire human brain. The experimental encephalitis model in Lewis rats was used to define affected cell populations after infection with the neurotropic MV strain CAM/RB. Distribution patterns of MV were analysed by appropriate cell markers in the brain sections of infected animals employing multiple immunofluorescence labelling and confocal laser scanning microscopy. MV was detected in neurones but not in astrocytes, oligodendrocytes, microglia, and endothelial cells. GABAergic and glutamatergic neurons displayed MV antigen whereas cholinergic and catecholaminergic neurons appeared devoid of MV immunoreactivity. Mapping of the rat brain has revealed MV-infected neurones predominantly in motor, somatosensory, auditory, and visual cortices as well as in the basal ganglia and thalamic nuclei of infected rats. The results indicate that MV apparently disseminates via GABAergic and glutaminergic neurones and their processes. The tightly restricted viral distribution pattern is consistent with both inefficient immune clearance from infected neurones and with the observed disease symptoms.