FPCountR: Absolute quantification of fluorescent proteins for synthetic biology

This paper presents a generalisable method for the calibration of fluorescence readings on microplate readers, in order to convert arbitrary fluorescence units into absolute units. FPCountR relies on the generation of bespoke fluorescent protein (FP) calibrants, assays to determine protein concentration and activity, and a corresponding analytical workflow. We systematically characterise the assay protocols for accuracy, sensitivity and simplicity, and describe a novel ‘ECmax’ assay that outperforms the others and even enables accurate calibration without requiring the purification of FPs. To obtain cellular protein concentrations, we consider methods for the conversion of optical density to either cell counts or alternatively to cell volumes, as well as examining how cells can interfere with protein counting via fluorescence quenching, which we quantify and correct for the first time. Calibration across different instruments, disparate filter sets and mismatched gains is demonstrated to yield equivalent results. It can also reveal that mCherry absorption at 600nm does not confound cell density measurements unless expressed to over 100,000 proteins per cell. FPCountR is presented as pair of open access tools (protocol and R package) to enable the community to use this method, and ultimately to facilitate the quantitative characterisation of synthetic microbial circuits.

Quantitative characterisation of low abundant yeast mitochondrial proteins reveals compensation for haplo-insufficiency in different environments

Quantification of low abundant membrane-bound proteins such as transcriptional factors and chaperones has been proved difficult even with the most sophisticated analytical technologies. Here we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for quantitation of low abundance protein and as proof of principle we choose two interacting membrane-bound proteins involved in fission of mitochondria in yeast. In Saccharomyces cerevisiae the recruitment of Fis1p and Mdv1p fission proteins to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We used FCS in single, GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells, and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism which controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.

Quantitative characterisation of extended-release tablets with quetiapine using NIR-chemometric methods

This study aims to develop and validate NIR-chemometric methods for quantifying the API (quetiapine) and two excipients in extended-release tablets without sample preparation. The calibration samples were prepared following an experimental design with three variables (quetiapine, HPMC and microcrystalline cellulose) and five levels (concentration 80-90-100-110-120% of API). The validation set included three concentration levels (90-100-110%). The best calibration algorithms have used the same pre-treatment method (SNV), and different factors: 7 PLS factors (R² -0,966 and RMSEP-6,84) for quetiapine, 8 PLS factors (R²-0,927 and RMSEP 6,84) for HPMC and 3 PLS factors (R²-0,983 and RMSEP-7,26) for microcrystalline cellulose. The methods were fully validated according to the ICH guidance using these calibration models. Regarding the trueness of the methods, the recovery was between 98.51 and 99.43 for quetiapine, between 98.61 and 100.85 for HPMC, and between 100.61 and 101.78 for microcrystalline cellulose. According to data obtained, the accuracy profile was ± 5 for quetiapine and HPMC, and ± 6 for microcrystalline cellulose. Linearity profile was also in establish intervals at accuracy and the R2 value was 0.983 for quetiapine, 0.948 for HPMC and 0.997 for microcrystalline cellulose. In conclusion, the developed NIR-chemometric methods have suitable reproducibility, accuracy, linearity and can be used for quantitative characterisation of extended-release tablets with quetiapine, with any sample preparation.

Label-free three-dimensional observations and quantitative characterisation of on-chip vasculogenesis using optical diffraction tomography

Label-free, three-dimensional (3D) quantitative observations of on-chip vasculogenesis were achieved using optical diffraction tomography. Exploiting 3D refractive index maps as an intrinsic imaging contrast, the vascular structures, multicellular activities, and...

Amyloid particles facilitate surface-catalyzed cross-seeding by acting as promiscuous nanoparticles

ABSTRACTAmyloid seeds are nanometre-sized protein particles that accelerate amyloid assembly, as well as propagate and transmit the amyloid protein conformation associated with a wide range of protein misfolding diseases. However, seeded amyloid growth through templated elongation at fibril ends cannot explain the full range of molecular behaviours observed during cross-seeded formation of amyloid by heterologous seeds. Here, we demonstrate that amyloid seeds can accelerate amyloid formation via a surface catalysis mechanism without propagating the specific amyloid conformation associated with the seeds. This type of seeding mechanism is demonstrated through quantitative characterisation of the cross-seeded assembly reactions involving two non-homologous and unrelated proteins: the human Aβ42 peptide and the yeast prion-forming protein Sup35NM. Our results suggest experimental approaches to differentiate seeding by templated elongation from non-templated amyloid seeding, and rationalise the molecular mechanism of the cross-seeding phenomenon as a manifestation of the aberrant surface activities presented by amyloid seeds as nanoparticles.