Biology subjects

Hatton, D.

Publications and source records attributed to Hatton, D..

10 recordsLinked to original sources

bioRxiv2026

Automating 3DED data processing at eBIC

Three-dimensional electron diffraction (3DED) is an emerging and useful technique for solving molecular structures of small and biological macro-molecules from nanometre-sized crystals. We present our automated data processing workflow for 3DED datasets collected at Diamond Light Sources electron Bio-Imaging Centre (eBIC). For this purpose, we developed a package called AutoED. The processing pipeline includes data collection, analysis of the beam position, metadata gathering, file conversion, and finally data processing using xia2 (which supports both DIALS and XDS). The processing results are captured in a summary report produced by AutoED. Our main goal is to reduce the workload of electron diffraction scientists, but also to enforce good standards already used in macromolecular crystallography (MX). All the collected 3DED datasets are automatically converted into NeXus data format which is considered a Gold Standard for MX. This standardized data format allows for all the relevant metadata about the experiment to be kept together with diffraction images. We also discuss the methods used in AutoED to determine the electron beam position on diffraction images.

biophysics↗

bioRxiv2026

DNA Damage Driven Viability Loss and Transcriptional Reprogramming in Chinese Hamster Ovary Cell Perfusion Culture

Intensified perfusion cultures promise higher yields and consistent product quality, yet extended runs frequently stall due to declining Chinese hamster ovary (CHO) cell viability at high cell density. Here, we identify DNA damage accumulation as a central, previously underappreciated driver of this limitation. Using two antibody-expressing CHO cell lines operated at a control and high cell density in perfusion bioreactors, we combined time-resolved transcriptomics with molecular, biophysical and super-resolution imaging analyses. We observed a progressive, global downregulation of DNA damage response (DDR) pathways accompanied by a time-dependent accumulation of DNA lesions. Notably, {gamma}H2AX signalling declined despite DNA damage, indicating impaired damage sensing and repair. Concomitantly, RNA polymerase II protein levels and transcriptional hub organisation were markedly reduced, consistent with widespread transcriptional dysfunction preceding loss of viability. An extended 21-day perfusion run confirmed continued viability decline beyond day 14, supporting a cumulative damage model. Comparison with HEK293 cells revealed substantially more efficient DNA repair, underscoring an intrinsically attenuated DDR in CHO cells. Together, these findings demonstrate that unrepaired DNA damage fundamentally constrains perfusion culture longevity, exposing limits of CHO genomic plasticity and highlighting DDR pathways as promising targets for host cell engineering to enhance perfusion performance.

bioengineering↗

bioRxiv2025

Automated filtering of particle images in single particle cryoEM

Continued exponential growth in the number of structures resolved by single particle cryoEM, as seen in the last decade, requires ever more effective data analysis workflows. Datasets are rarely homogeneous, demanding a multistep procedure for discarding outliers. Since individual particles are very noisy, either 2D or 3D averages are normally used for discrimination. This becomes challenging when the 2D classes themselves are heterogeneous, leading to selection of contaminants or discarding useful rare views/poses. The 3D model-based discrimination requires trustworthy 3D maps and a correct assignment of Euler angles, which in turn depends on the quality of the initial data and might not be available at the very early stages of the analysis. We propose a novel deep-learning approach for improving quality of single particle datasets. The two-stage procedure consists of denoising single particle images using Variational AutoEncoder framework followed by particle quality filtering based on the score inferred for every particle by Domain Adaptation Neural Network trained on a large data set of categorised 2D averages. This approach allows an automated scoring of noisy raw images using data patterns learned from the high signal-to-noise ratio, externally derived 2D classes. Consequently, a higher quality data set enters computationally expensive steps of the data analysis, reducing the need for protracted and expensive calculations. Importantly, our method does not require any prior knowledge about the data or existence of a 3D model, making it universally applicable. Tests on publicly available datasets demonstrated that our approach largely outperformed 2D class-based particle discrimination. Smaller subsets of the top-scoring particles selected with our method were required to obtain the author-reported 3D model resolution. When applied to the user data in the automated on-the-fly data processing pipeline, the method rescued 30% of cases, which otherwise would not reach confidence threshold required for making decision to proceed to the 3D model refinement. It also led to general improvements in the quality of the 3D models for many datasets which were selected for the high-resolution processing.

biophysics↗

bioRxiv2024

Identification of Cellular Signatures Associated with Chinese Hamster Ovary (CHO) Cell Adaptation for Secretion of Antibodies

The secretory capacity of Chinese hamster ovary (CHO) cells remains a fundamental bottleneck in the manufacturing of protein-based therapeutics. Unconventional biological drugs with complex structures and processing requirements are particularly problematic. Although engineered vector DNA elements can achieve rapid and high-level therapeutic protein production, a high metabolic and protein folding burden is imposed on the host cell. Cellular adaptations to these conditions include differential gene expression profiles that can in turn influence the productivity and quality control of recombinant proteins. In this study, we used quantitative transcriptomics and proteomics analyses to investigate how biological pathways change with antibody titre. Gene and protein expression profiles of CHO pools and clones producing a panel of different monoclonal and bispecific antibodies were analysed during fed-batch production. Antibody-expressing CHO pools were heterogeneous, resulting in few discernible genetic signatures. Clonal lines derived from these pools, selected for high and low production, yielded a small number of differentially expressed proteins that correlated with productivity and were shared across biotherapeutics. However, the dominant feature associated with higher protein production was transgene copy number and resulting mRNA expression level. Moreover, variability between clones suggested that the process of cellular adaptation is variable with diverse cellular changes associated with individual adaptation events.

bioengineering↗

bioRxiv2024

EMinsight: a tool to capture cryoEM microscope configuration and experimental outcomes for analysis and deposition.

The widespread adoption of cryoEM technologies for structural biology has pushed the discipline to new frontiers. A significant worldwide effort has refined the Single Particle Analysis (SPA) workflow into a reasonably standardised procedure. Significant investment of development time have been made particularly in sample preparation, microscope data collection efficiency, pipeline analyses and data archiving. The widespread adoption of specific commercial microscopes, software for controlling them and best practises developed at national facilities has also begun to establish a degree of standardisation to data structures coming from the SPA workflow. There is opportunity to capitalise on this moment in the fields maturation, to capture metadata from SPA experiments and correlate this with experimental outcomes, which is presented here in a set of programmes called EMinsight. This tool aims to prototype the framework and types of analyses that could lead to new insights into optimal microscope configurations as well as for defining methods for metadata capture to assist with archiving of cryoEM SPA data. We also envisage this tool to be useful to microscope operators and facilities looking to rapidly generate reports on SPA data collection and screening sessions. SynopsisEMinsight is a Python-based tool for systematically mining metadata from single particle analysis cryoEM experiments. The capture and analysis of metadata facilitates assessment of instrument performance, provides concise reporting of experiment performance and sample quality by analysing preprocessing results, and gathers metadata for deposition. We envisage this approach to benefit the microscope operator, facility managers, database developers and users.

biophysics↗

bioRxiv2022

Deciphering the determinants of recombinant protein yield across the human secretome

Mammalian cells are critical hosts for the production of most therapeutic proteins and many proteins for biomedical research. While cell line engineering and bioprocess optimization have yielded high protein titers of some recombinant proteins, many proteins remain difficult to express. Here, we decipher the factors influencing yields in Chinese hamster ovary (CHO) cells as they produce 2165 different proteins from the human secretome. We demonstrate that variation within our panel of proteins cannot be explained by transgene mRNA abundance. Analyzing the expression of the 2165 human proteins with machine learning, we find that protein features account for only 15% of the variability in recombinant protein yield. Meanwhile, transcriptomic signatures account for 75% of the variability across 95 representative samples. In particular, we observe divergent signatures regarding ER stress and metabolism among the panel of cultures expressing different recombinant proteins. Thus, our study unravels the factors underlying the variation on recombinant protein production in CHO and highlights transcriptomics signatures that could guide the rational design of CHO cell systems tailored to specific proteins.

systems biology↗

bioRxiv2022

The actin nucleator Spire cooperates with Rab11 to initiate nascent dendrite branches.

Dendrites form arbors whose size, shape and complexity define how neurons cover their receptive territories. Actin dynamics contribute to growth and remodeling of dendrite arbors. Here we examined how Spire, a conserved actin nucleation factor, promotes formation of new branches in vivo. In live imaging of Drosophila class IV dendritic arborization (c4da) neurons, Spire was observed at new sites of branch initiation, where it assembled new actin polymer in a burst just prior to filopodial outgrowth. For dendrite arborization, Spire required intact structural domains to nucleate actin and target the secretory network, and interacted with Rab11 GTPase, a key regulator of recycling endosomes. Together, these findings support a model in which Spire cooperates with Rab11 to promote new dendrite branches by linking localized actin dynamics with intracellular trafficking of endosomes that deliver lipids and cargoes to fuel protrusive outgrowth of nascent dendrites.

neuroscience↗

bioRxiv2021

Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.

systems biology↗

bioRxiv2020

Transcriptome analysis of EPO and GFP HEK293 Cell-lines Reveal Shifts in Energy and ER Capacity Support Improved Erythropoietin Production in HEK293F Cells

Higher eukaryotic cell lines like HEK293 are the preferred hosts for production of therapeutic proteins requiring human post translational processing. However, recombinant protein production can result in severe stress on the cellular machinery, resulting in limited titre and product quality. To investigate the cellular and metabolic characteristics associated with these limitations, we compared erythropoietin (secretory) and GFP (non-secretory) protein producer HEK293 cell-lines using transcriptomics analysis. Despite the high demand for ATP in all protein producer clones, a significantly higher capacity for ATP production was observed with erythropoietin producers as evidenced by the enrichment of upregulated genes in the oxidative phosphorylation pathway. In addition, ribosomal genes exhibited specific patterns of expression depending on the recombinant protein and the production rate. In a clone displaying a dramatically increased erythropoietin secretion, we detected higher ER stress, including upregulation of the ATF6B gene. Our results are significant in recognizing key pathways for recombinant protein production and identifying potential target genes for further development of secretory power in mammalian cell factories. In BriefAlthough the protein secretion process has been widely studied, the complexity of it leaves many questions with regards to defining bottlenecks for successful protein secretion to be answered. By investigating the transcriptomic profiles of different HEK293 clones with varying translational rates producing either the secreted protein erythropoietin or the intracellular GFP, we reveal that high ATP production and improved capacity of specific post-translational pathways are key factors associated with boosting erythropoietin production. HighlightsO_LITranscriptomics analysis of a panel of HEK293 stable cell lines expressing GFP or erythropoietin (EPO) at varying translational rates C_LIO_LIExpression of mitochondrial ribosomal genes is positively correlated with EPO secretion C_LIO_LIExpression of different cytosolic ribosomal genes are correlated with productivity in a recombinant-protein specific manner C_LIO_LIHigh EPO producing clones have significant upregulation of ATF6B, potentially enabling a beneficial ER stress response to cope with high protein secretion C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/299966v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@1e35efforg.highwire.dtl.DTLVardef@71cee2org.highwire.dtl.DTLVardef@1c56d33org.highwire.dtl.DTLVardef@843a6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2020

Evolution from adherent to suspension - systems biology of HEK293 cell line development

The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production and is today used for manufacturing of several therapeutic proteins and viral vectors. Despite this increasing popularity there is still limited knowledge of the detailed genetic and composition of derivatives of this strain. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.

systems biology↗