Biology subjects

Gooding, J.

Publications and source records attributed to Gooding, J..

4 recordsLinked to original sources

bioRxiv2025

P3ANUT: An enhanced DNA sequencing analysis platform for uncovering and correcting errors in peptide phage display library screening

Display technologies are used extensively in the discovery of peptides and antibodies towards the development of new medicines and diagnostic tools. Phage display technology enables the filtering of <1010 random peptides/antibodies down to and enriched pool of candidates with favourable binding affinities. In recent years, next-generation DNA sequencing technologies have increased the precision and accuracy with which the peptide sequences of phage display library clones are identified. Inaccuracies in DNA sequencing such as substitutions, insertions and deletions in the library oligonucleotide region have the potential to result in the identification of erroneous candidate sequences. Here, we describe a Python Pipeline for Phage Analysis through a Normative Unified Toolset (P3ANUT) which employs Levenshtein distance, k-mer approaches and a novel encoding scheme on paired-end sequencing outputs to correct sequencing errors from next-generation sequencing outputs of display library screens. We introduce an easy-to-use and highly customisable computational tool with graphical user- and command line interfaces to process entire datasets within a single input, as well as visualisation tools for candidate analysis and data generation. P3ANUT shows significant improvements in read recovery, overall read quality, and runtime compared to a previously published pipeline. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=96 SRC="FIGDIR/small/648809v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@15e1172org.highwire.dtl.DTLVardef@cb9097org.highwire.dtl.DTLVardef@81cabcorg.highwire.dtl.DTLVardef@1253865_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Electrochemically controlled switching of dyes for enhanced super-resolution optical fluctuation imaging (SOFI)

In super-resolution optical fluctuation imaging (SOFI), the locations of molecules spaced closer than the diffraction limit of light can be identified through spatial and temporal correlation analysis of the fluorescence intensity fluctuation. Using organic dyes as fluorophore probes, the fast but stochastic switching of the individual dyes is favourable for improving SOFI imaging resolution and speed, especially in the case of high-order cumulant analysis. While in practice, fluorophore switching can be non-uniform, with some fluorophores remaining in ON or OFF state for extended periods. Furthermore, in some cases the overall rate of switching of the fluorophores can be too slow, presenting practical limitations for high-resolution and fast SOFI imaging. In this work, we demonstrate how to overcome these challenges using electrochemical controlled fluorophore switching. The oscillating electrochemical potential setting with high frequency increases the switching rate and reduces the switching heterogeneity of fluorophores. The dye Alexa Fluor 647, applied here as an example, exhibits over 3-fold decrease in average ON time and over 2-fold decrease in switching variance, resulting in significantly improved SOFI image resolution with fewer frames. We demonstrated that this new electrochemically controlled SOFI imaging modality can achieve a SOFI image with [~]130 nm resolution in 2 seconds of acquisition time, and 80 nm resolution in 6 seconds. This advancement enables fast, large area tile-scan super-resolution imaging, which opens the full potential of SOFI imaging.

biophysics↗

bioRxiv2020

Evaluation of optically tailored fluorescent silicon quantum dots for bioimaging of the tear film

This experimental study aimed to investigate the feasibility of using silicon quantum dots doped with transition metals: scandium, copper and zinc as contrast agents for eventual application for the study of the tear film in eyes. Si-QDs were synthesized and characterized by transmission electron microscopy, photoluminescence, absorbance and transient absorption measurements. The fluorescence of Si-QDs was investigated when combined with TheraTears(R) (a balanced electrolyte formula for dry eye therapy). An optical imaging system composed of a modified slit lamp biomicroscope combined with a high-resolution Zyla sCMOS camera, SOLIS software, custom-made optical mounts and emission filters (460 nm, 510 nm and 530 nm) were used for in vitro imaging of Si-QDs with TheraTears(R). The average size of Si-QDs was 2.65 nm. In vitro imaging of Sc-Si-QDs and Cu-Si-QDs indicated their stable and bright fluorescence with TheraTears(R). Sc-Si-QDs were significantly brighter compared to Cu-Si-QDs and Zn-Si-QDs, and the Zn-Si-QDs showed a tendency to clump in TheraTears(R). The fluorescence of the Si-QDs was detected down to a concentration of 0.01 {micro}g/mL within a total volume of 5 {micro}L. Cu-Si-QDs and Sc-Si-QDs showed brighter fluorescence than Zn-Si-QDs. However, Zn-Si-QDs and to a lesser extent, Cu-Si-QDs showed some aggregation at specific concentrations. Sc-Si-QDs are proposed as a better option for further development as an in vivo bioimaging agent to study the tear film dynamics.

biochemistry↗

bioRxiv2020

Precise, high-throughput production of multicellular spheroids with a bespoke 3D bioprinter

3D in vitro cancer models are important therapeutic and biological discovery tools, yet formation of multicellular spheroids in a throughput and highly controlled manner to achieve robust and statistically relevant data, remains challenging. Here, we developed an enabling technology consisting of a bespoke drop-on-demand 3D bioprinter capable of high-throughput printing of 96-well plates of spheroids. 3D-multicellular spheroids are embedded inside a tissue-like matrix with precise control over size and cell number. Application of 3D bioprinting for high-throughput drug screening was demonstrated with doxorubicin. Measurements showed that IC50 values were sensitive to spheroid size, embedding and how spheroids conform to the embedding, revealing parameters shaping biological responses in these models. Our study demonstrates the potential of 3D bioprinting as a robust high-throughput platform to screen biological and therapeutic parameters. Significance StatementIn vitro 3D cell cultures serve as more realistic models, compared to 2D cell culture, for understanding diverse biology and for drug discovery. Preparing 3D cell cultures with defined parameters is challenging, with significant failure rates when embedding 3D multicellular spheroids into extracellular mimics. Here, we report a new 3D bioprinter we developed in conjunction with bioinks to allow 3D-multicellular spheroids to be produced in a high-throughput manner. High-throughput production of embedded multicellular spheroids allowed entire drug-dose responses to be performed in 96-well plate format with statistically relevant numbers of data points. We have deconvoluted important parameters in drug responses including the impact of spheroid size and embedding in an extracellular matrix mimic on IC50 values.

bioengineering↗