Biology subjects

Clarke, D.

Publications and source records attributed to Clarke, D..

3 recordsLinked to original sources

bioRxiv2018

Development of novel lipidic particles for siRNA delivery that are highly effective after 12 months storage

Liposomes are versatile and well-proven as a means to deliver nucleic acids into cells. Most of the formulation procedures used are labour intensive and result in unstable end products. We have previously reported on the development of a simple, yet efficient, hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles. Here we show that the particles are stable up to 12 months after storage room temperature (RT), 4{degrees}C or - 20{degrees}C. While RT storage results in changes in particle size and polydispersity, gene silencing of all particles was similar to freshly prepared particles following storage for 3, 6, 9 or 12 months at all temperatures. This is the first report of such long-term stability in siRNA-loaded liposomes.

biochemistry↗

bioRxiv2018

Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve <8 nm localisation precision and 12 nm resolution under cryogenic conditions in a low-cost, low-tech system. This is to our knowledge the best resolution yet attained in biological samples. Furthermore, we demonstrate multicolour imaging and show that the inexpensive setup outperforms 10-fold more costly super-resolution microscopes. By also removing the barrier to total internal reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

biophysics↗

bioRxiv2018

Characterization of the role of glycine lipids in Bacteroides thetaiotaomicron

Acylated amino acids function as important components of the cellular membrane in some bacteria. Biosynthesis is initiated by the N-acylation of the amino acid and this is followed by subsequent O-acylation of the acylated molecule resulting in the production of the mature diacylated amino acid lipid. In this study we use both genetics and liquid chromatography-mass spectrometry (LC-MS) to characterize the biosynthesis and function of novel diacylated glycine lipid (GL) species in Bacteroides thetaiotaomicron. We, and others, have previously reported the identification of a gene, named glsB in this study, that encodes a N-acyltransferase activity responsible for the production of a monoacylated glycine called N-acyl-3-hydroxy-palmitoyl glycine (or commendamide). In all of the Bacteroidales genomes so far sequenced the glsB gene is located immediately downstream from a gene, named glsA, also predicted to encode a protein with acyltransferase activity. We use LC-MS to show that co-expression of glsB and glsA results in the production of GL in Escherichia coli. We constructed a deletion mutant of the glsB gene in B. thetaiotaomicron and we confirm that glsB is required for the production of GL in B. thetaiotaomicron. Moreover, we show that glsB is important for the ability of B. thetaiotaomicron to adapt to stress and colonize the mammalian gut. Therefore, this report is the first to describe the genetic requirements for the biosynthesis of GL, a novel diacylated amino acids species that contributes to fitness in the human gut bacterium, B. thetaiotaomicron.

microbiology↗