Multimodal binding and inhibition of bacterial ribosomes by the antimicrobial peptides Api137 and Api88.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH2) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

Cryo-EM captures early ribosome assembly in action.

Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.

Visualization of Cortical Modules in Flattened Mammalian Cortices.

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

Sexually selected size differences and conserved sexual monomorphism of genital cortex.

The mammalian somatosensory cortex shows marked species-specific differences. How evolution in general and sexual selection in particular shape the somatosensory cortical body representation has not been delineated, however. Here we address this issue by a comparative analysis of genital cortex. Genitals are unique body parts in that they show sexual dimorphism, major changes in puberty and typically more pronounced species differences than other body parts (Hosken & Stockley, 2004). To study the evolution of genital cortex we flattened cortical hemispheres and assembled 104 complete body maps, revealed by cytochrome-oxidase activity in layer 4 of 8 rodent and 1 lagomorph species. In two species, we also performed antibody stainings against vesicular glutamate transporter-2, which suggested that cytochrome-oxidase maps closely mirror thalamic innervation. We consistently observed a protrusion between hindlimb and forelimb representation, which in rats (Lenschow et al., 2016) corresponds to the penis representation in males and the clitoris representation in females. Consistent with the idea that this protrusion corresponds to genital cortex, we observed a size increase of this protrusion during puberty. Species differed in external genital sexual dimorphism, but we observed a sexual monomorphism of the putative genital protrusion in all species, similar to previous observations in rats. The relative size of the putative genital protrusion varied more than 3-fold between species ranging from 0.5% of somatosensory cortex area in chipmunks to 1.7% in rats. This relative size of the genital protrusion co-varied with relative testicle size, an indicator of sperm competition and sexual selection.