Biology subjects

Barran, P.

Publications and source records attributed to Barran, P..

9 recordsLinked to original sources

bioRxiv2026

Fatty Acid β-oxidation and Ferroptosis Define a Survival-Productivity Trade Off in CHO fed-Batch Bioreactors

Fed-batch production in Chinese hamster ovary cells is usually optimised empirically, yet the intracellular mechanisms that determine whether cells sustain productivity or enter terminal decline remain poorly resolved. Here, we combine longitudinal proteomics and intracellular metabolomics across differential ambr250 fed-batch processes to define the metabolic programmes associated with CHO cell viability and antibody production. Across media/feed combinations and an intensified seeding regime, culture progression followed a conserved trajectory from proliferation to metabolic transition and terminal stress. The high-stress state was characterised by a switch toward mitochondrial and peroxisomal fatty acid {beta}-oxidation, lipid remodelling, oxidative burden and activation of ferroptosis-associated pathways. Enriched feed conditions delayed this transition through enhanced redox and glutathione-linked defence, but did not proportionally increase antibody titre, revealing a trade-off in which cellular resources are diverted from recombinant protein production toward survival. These data identify fatty acid metabolism and ferroptosis as key constraints on late-stage CHO fed-batch performance and provide a mechanistic framework for rational feed design and host-cell engineering.

cell biology↗

bioRxiv2026

Label-Free Determination of Chondroitin Sulphate from Microgram Quantities of Human Milk

Human milk contains structurally diverse glycans with key roles in shaping infant development, yet analytical constraints limit characterisation from low-volume samples. Glycosaminoglycans (GAGs), including chondroitin sulphate (CS), are understudied due to existing protocols requiring sample volumes of at least 5 mL and lengthy extraction steps prior to instrumental analysis. This study establishes a workflow for quantifying CS disaccharides from 25 {micro}L of human milk, enabling analysis of samples previously inaccessible to GAG profiling, such as those collected as salvage samples from neonatal intensive care units. For CS quantification, the CS is first enzymatically depolymerised using chondroitinase ABC to release repeating disaccharide units. Matrix complexity is reduced via two rounds of acetonitrile-based protein and lipid precipitation. Disaccharides are separated by hydrophilic interaction liquid chromatography and detected using a Triple Quadrupole Mass Spectrometer, providing robust sensitivity for all CS disaccharides. Method development and validation were performed using pooled mature human milk from term infants. This workflow facilitates detection of all CS disaccharides, with low but reproducible recoveries for total CS. Low- and high-level spike recoveries were 41.3% (RSDr 7.5%, RSDiR 15.9%) and 43.7% (RSDr 24.4%, RSDiR 27.9%), respectively. Despite modest absolute accuracy, precision remained sufficient to make relative comparison of CS concentrations between samples. This method expands the analytical toolkit for human milk glycomics, enabling same day preparation and CS profiling from sample volumes that are 200 times smaller than prior work, supporting future investigations into GAG-mediated functions in early life. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723732v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@21a79borg.highwire.dtl.DTLVardef@182dc43org.highwire.dtl.DTLVardef@4aeae0org.highwire.dtl.DTLVardef@822dd1_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Schematic of sample preparation protocol 25 L of human milk is combined with lyase enzymes and TRIS buffer containing the internal standard prior to incubation. Samples then undergo multiple rounds of centrifugation and refrigeration before analysis via LC-MS/MS. Made using BioRender.com. Glycan nomenclature following Varki et al., 2015. C_FIG

biochemistry↗

bioRxiv2025

From Discovery to Process Control: Positioning DIA Proteomics in Biomanufacturing Pipelines

Gaining control of existing biomanufacturing chassis organisms, such as Escherichia coli K12, and novel isolates, such as the salt tolerant Halomonas bluephagenesis sp TD01 studied here may be facilitated by the investigation and monitoring of their metabolic and regulatory processes, particularly through proteomics. Here we consider the performance of a range of typically available proteomics platforms across a range of price points to map chassis organisms metabolic pathways. A set of model bacterial samples was prepared from E. coli and H. bluephagenesis sp. TD01 in 1:1, 1:2 and 2:1 ratios and analyzed using five LC-MS systems. Of the 8,222 proteins identified across all samples analysed (4,401 proteins from E. coli; 3,821 from Halomonas sp. TD01), the TimsTOF and Exploris were able to achieve extensive proteome coverage quantifying 5.5k and 5k proteins respectively, with the ZenoTOF, Waters MRT and the legacy Waters Vion respectively quantifying 3.5k, 1.3k, and [~]850 proteins at 1% FDR. Proteins comprising core metabolic pathways critical to biomanufacturing in these chassis organisms can be quantified with all instruments. We characterize metabolic adaptation in H. bluephagenesis by showing that replacement of glucose with a carboxylic acid feed stock directs carbon flux towards potential butane precursors as well as how the acquired data permits monitoring of the cobalamin (vitamin B12) production pathway. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/695173v3_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@148fd14org.highwire.dtl.DTLVardef@df4b7borg.highwire.dtl.DTLVardef@1d479b6org.highwire.dtl.DTLVardef@82a4df_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Investigation of Volatile Metabolites in Sebum as Prodromal Indicators of Parkinson's Disease

Parkinsons Disease (PD) has been associated with a distinct odour, which emanates from the skin and is strongest in sebum-rich areas. In this study, sebum was sampled from participants using cotton gauze and the volatile components emanating from these swabs were analysed directly with thermal desorption gas chromatography - mass spectrometry (TD GC-MS). We analysed subjects with clinically established PD (n=46) along with healthy controls (n=28) sampled from two sites. The volatilome profiles obtained for PD and control cohorts were compared with the profile of participants (n=9) with isolated REM sleep behaviour disorder (iRBD) to investigate metabolite changes in probable prodromal PD. We also compared PD participants sampled at yearly intervals for a total of three years. Volatile compounds from TD GC-MS analysis were found in different quantities between PD, control and iRBD subjects. We found 55 significant features where abundance in samples from individuals with iRBD was intermediate between that found for PD and control samples. Significant features were found to be alkanes and fatty acid methyl esters (FAMEs), with other metabolites identified as an aldehyde, purine and tropinone. In olfactory analysis of the iRBD samples three out of nine were classified PD, and on clinical follow up two of these showed PD symptoms. Further, when analysing the volatilome from longitudinal PD sampling, almost two-thirds of the significant features showed differential regulation over the three visits. Our findings support the use of sebum as an accessible biofluid rich with measurable volatile compounds which alter in abundance in individuals with PD and iRBD, as the disease progresses.

neuroscience↗

bioRxiv2022

Ion mobility mass spectrometry and molecular dynamics simulations unravel the conformational stability and coordination dynamics of human metallothionein-2 species

Ion mobility-mass spectrometry (IM-MS) unraveled different conformational stability in Zn4-7-metallothionein-2. We introduced a new molecular dynamics simulations approach that permitted the exploration of all conformational space confirming the experimental data, and revealed that not only the Zn-S bonds but also -{beta} domain interaction modulates protein unfolding.

biochemistry↗

bioRxiv2021

Novel allosteric mechanism of dual p53/MDM2 and p53/MDM4 inhibition by a small molecule

Restoration of the p53 tumor suppressor for personalised cancer therapy is a promising strategy. However, high-affinity MDM2 inhibitors have shown substantial side effects in clinical trials. Thus, elucidation of the molecular mechanisms of action of p53 reactivating molecules with alternative functional principle is of the utmost importance. Here, we report a discovery of a novel allosteric mechanism of p53 reactivation through targeting the p53 N-terminus which blocks both p53/MDM2 and p53/MDM4 interactions. Using biochemical assays and molecular docking, we identified the binding site of two p53 reactivating molecules, RITA and protoporphyrin IX (PpIX). Ion-mobility mass spectrometry revealed that the binding of RITA to serine 33 and serine 37 is responsible for inducing the allosteric shift in p53, which shields the MDM2 binding residues of p53 and prevents its interactions with MDM2 and MDM4. Our results point to an alternative mechanism of blocking p53 interaction with MDM2 and MDM4 and may pave the way for the development of novel allosteric inhibitors of p53/MDM2 and p53/MDM4 interactions. Contribution to the fieldGiven the immense importance of the p53 tumor suppressor for cancer, efforts have been made to identify p53 activators, which sterically inhibit MDM2. Because high-affinity MDM2 inhibitors are facing problems with considerable side effects, other approaches are needed to reactivate p53 for improved cancer therapy. The allosteric mechanism of action of p53 activator RITA, which we discovered, and its dependence on the oncogenic switch, is an unexpected turn in the p53 story. Our findings provide a basis for the development of p53 activators with a similar mode of functioning, either through the classical drug discovery route or through the drug repurposing approach. Allosteric modulators might have great potential as single agents, or in combination with the standard of care. Further, p53 modulators could serve as invaluable tools to better understand its biology.

biochemistry↗

bioRxiv2018

Discovery of volatile biomarkers of Parkinsons disease from sebum

Parkinsons disease (PD) is a progressive, neurodegenerative disease that presents with significant motor symptoms, for which there is no diagnostic test (1-3). We have serendipitously identified a hyperosmic individual, a Super Smeller that can detect PD by odor alone, and our early pilot studies have indicated that the odor was present in the sebum from the skin of PD subjects(4). Here, we have employed an unbiased approach to investigate the volatile metabolites of sebum samples obtained non-invasively from the upper back of 64 participants in total (21 controls and 43 PD subjects). Our results, validated by an independent cohort, identified a distinct volatiles-associated signature of PD, including altered levels of perillic aldehyde and eicosane, the smell of which was then described as being highly similar to the scent of PD by our Super Smeller.\n\n1 sentence summaryMetabolomics identifies volatile odorous compounds from patient sebum that associate with the smell of Parkinsons.

neuroscience↗

bioRxiv2018

Native Mass Spectrometry Reveals the Conformational Diversity of the UVR8 Photoreceptor

UVR8 is a plant photoreceptor protein that regulates photomorphogenic and protective responses to UV light. The inactive, homodimeric state absorbs UV-B light resulting in dissociation into monomers, which are considered to be the active state and comprise a {beta}-propeller core domain and intrinsically disordered N- and C-terminal tails. The C-terminus is required for functional binding to signalling partner COP1. To date, however, structural studies have only been conducted with the core domain where the terminal tails have been truncated. Here, we report structural investigations of full-length UVR8 using native ion mobility mass spectrometry adapted for photo-activation. We show that, whilst truncated UVR8 photo-converts from a single conformation of dimers to a single monomer conformation, the full-length protein exist in numerous conformational families. The full-length dimer adopts both a compact state and an extended state where the C-terminus is primed for activation. In the monomer the extended C-terminus destabilises the core domain to produce highly extended yet stable conformations, which we propose are the fully active states that bind COP1. Our results reveal the conformational diversity of full-length UVR8. We also demonstrate the potential power of native mass spectrometry to probe functionally important structural dynamics of photoreceptor proteins throughout nature.\n\nTOC Graphic\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC=\"FIGDIR/small/371658_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (60K):\norg.highwire.dtl.DTLVardef@177c6f9org.highwire.dtl.DTLVardef@a845c9org.highwire.dtl.DTLVardef@17db14forg.highwire.dtl.DTLVardef@103d4f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2017

ORIGAMI: A Software Suite for Activated Ion Mobility Mass Spectrometry (aIM-MS) Applied To Multimeric Protein Assemblies

We present here a software suite (ORIGAMI) that facilitates the rapid acquisition and analysis of ion mobility data following collisional activation. ORIGAMI was developed for use on Waters Synapt instruments where data acquisition is achieved by interfacing WREnS (Waters Research Enabled Software) and MassLynx. Two components are presented, the first is ORIGAMIMS which enables activation of ions by sequential increase of collision voltages prior to ion mobility analysis. We demonstrate the use of ORIGAMI on the tetrameric assemblies formed by the proteins concanavalin A (103 kDa) and alcohol dehydrogenase (143 kDa). Activation is performed in the trap collision cell of the Synapt TriWave assembly, where the collision voltage can be ramped from 0-200 V. All of the acquired data is recorded in a single file which simplifies data acquisition. This substantially decreases the time needed to perform a typical activated IM-MS experiment on a single protein charge state from approx. 2 hours to ~25 minutes. Following data acquisition the data is analysed in the second component, ORIGAMIANALYSE, which allows the user to visualise the effect of activation on the mobility of the parent ion, as well as on any produced fragment ion. The user can export the data in the form of heat maps, waterfall or wire plots. In addition, tools implemented in ORIGAMI enable easy data extraction from single or multiple MassLynx .raw files, in-depth interrogation of large datasets, statistical analysis and figure creation capabilities. We demonstrate the use of ORIGAMI on concanavalin A and alcohol dehydrogenase acquired using the traditional protocols and the ORIGAMIMS method.\n\nHighlights- New methodology for faster acquisition and data analysis following activation of ions separated by ion mobility mass spectrometry\n- Software package capable of simultaneous analysis of multiple MassLynx .raw files\n- Visualization of the change in mobility of parent and fragment ions following activation\n- Easy extraction, data processing and extensive plotting tools

bioinformatics↗