Biology subjects

Hamilton, B. R.

Publications and source records attributed to Hamilton, B. R..

3 recordsLinked to original sources

bioRxiv2023

Effective concentration of marine nanoflagellates with a microfluidic device

Protist cells are typically manipulated through either centrifugation or membrane filtration, which can damage these fragile cell types. Use of microfluidic devices could greatly aid in the separation and concentration of protist cells with significantly less damage. Recent developments have enabled passive cell separation and consequent concentration based only on cell size. We utilize these advances to show that a passive spiral microfluidic device can effectively concentrate marine nanoflagellates within the 3-20 micron size range without harm to cells, while reducing background bacteria levels. The ability to concentrate these cell types appears only dependent on cell size, despite complicated cell surface geometries and motility. We anticipate that this approach will greatly aid researchers who require an ability to manipulate fragile cell types as well as reduce bacteria concentrations for experimental setups and cell isolation.

microbiology↗

bioRxiv2022

Genomic, Functional and Structural Analyses Reveal Mechanisms of Evolutionary Innovation within the Sea Anemone 8 Toxin Family

ShK from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. We identified ten SA8 genes in two clusters and six SA8 genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Our results provide the first demonstration that SA8 is a unique gene family in actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

genetics↗

bioRxiv2022

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery--either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Such enzymatic methods require careful optimisation of cyclisation over polymerisation. Here, we describe the engineering of self-cyclising "autocyclase" proteins, where an intramolecular rearrangement can be triggered to yield a monomeric cyclic product in high yields. We characterise the self-cyclisation reaction mechanism and demonstrate how the unimolecular reaction path can circumvent existing challenges of enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple and scalable way to access a vast diversity of macrocyclic biomolecules.

biochemistry↗