Biology subjects

Bose, D. A.

Publications and source records attributed to Bose, D. A..

5 recordsLinked to original sources

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↗

bioRxiv2026

A universal protein ladder for standardisation of diverse FRET assays

Fluorescence resonance energy transfer (FRET) is the highly distance dependent (3-10 nm) transfer of energy from a donor to an acceptor fluorophore, with transfer efficiency inversely proportional to the distance between the fluorophores. Consequently FRET serves as a powerful spectroscopic ruler for probing molecular interactions. Whilst cell based FRET assays report bulk relative changes in FRET efficiency in a population, single molecule FRET (smFRET) is capable of deconvoluting these population averages into distinct structural states. However, the lack of universal benchmarks prevents the direct translation of in vitro distance measurements to the intracellular environment and vice versa. Here, we present a modular protein ladder designed to harmonize FRET data across diverse platforms. Using an engineered repeating TPR motif and self-labeling enzymes, we demonstrate that our standards yield consistent FRET efficiencies across expression systems (mammalian and bacterial) and labelling strategies (self labelling enzymes and click chemistry with non-canonical amino acids). By providing a predictable calibration curve, the ladder enables interpolation between different experimental FRET modalities, including confocal smFRET, flow cytometry based-FRET and Fluorescence Lifetime Imaging Microscopy FRET (FLIM-FRET). This is the necessary infrastructure to relate molecular distances from the test tube to the cell.

biophysics↗

bioRxiv2025

Amyotrophic Lateral Sclerosis-associated 3' UTR enhancer embedded within CAV1 risk gene

Enhancer elements that reside within 3' untranslated regions (UTRs) are an understudied phenomenon. Given the independent regulatory functions of enhancers and 3' UTRs - enhancers governing pre-transcriptional control of gene expression and 3' UTRs mediating post-transcriptional regulation of messenger RNA (mRNA) fate - 3' UTR-associated enhancers may integrate these complementary layers to coordinate gene expression across multiple regulatory stages. Non-coding variation, impacting regulatory DNA, underpins the genetic architecture of disease. Indeed, the vast majority of single nucleotide polymorphisms (SNPs) associated with human complex diseases map to the non-coding genome, with causal variants particularly enriched within enhancers. Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder associated with non-coding risk variants, many of which are increasingly linked to enhancer disruption. The CAV1 gene, encoding the neuroprotective protein Caveolin-1, is a known ALS risk gene, yet the functional consequences of ALS-associated variation in its regulatory elements remain largely unexplored. Here, we combine genome-wide enhancer profiling with targeted experimental validation to define a previously uncharacterised ALS-associated enhancer embedded within the CAV1 3' UTR, systematically assess its regulatory potential, and evaluate the impact of ALS-associated SNPs on enhancer function. We show that an individual ALS-associated SNP within this 3' UTR-associated enhancer may disrupt function on multiple levels: at the DNA and chromatin level, by altering transcription factor binding with potential effects on recruitment of epigenetic co-regulators; and at the RNA level, by reshaping the structure and stability of a novel enhancer RNA transcribed from this locus. Collectively, our findings highlight this proximal CAV1/CAV2 enhancer as a functionally important regulatory element embedded with the 3' UTR of an ALS risk gene, illustrate how non-coding variants can impact multiple layers of gene regulation, and provide mechanistic insight into how intragenic enhancers contribute to ALS risk. More broadly, this work underscores the importance of 3' UTR-associated enhancers as modulators of risk gene expression and underexplored contributors to human complex disease.

genetics↗

bioRxiv2024

Cooperation between Intrinsically Disordered Regions regulates CBP condensate behaviour.

Intrinsically disordered regions (IDRs) have emerged as crucial regulators of protein function, allowing proteins to sense and respond to their environment. Creb binding protein (CBP) and EP300 (p300) are transcription coactivators that regulate gene expression in multicellular organisms, following their recruitment to cis-regulatory elements. CBP and p300 contain large IDRs, however little is known about how these different IDRs work together to regulate CBP function. Here, we show that CBP-IDRs cooperate to control different aspects of CBP behaviour in the nucleus, by regulating the properties of fluid-like condensates formed by endogenous CBP. We show how IDRs with different sequence properties make unique contributions to CBP behaviour by establishing a balance between positive and negative regulation of CBP condensates. These conflicting interactions are functionally important, shaping CBPs response to factors such as lysine acetylation, that influence condensate formation. When disrupted, regulatory CBP-IDRs change how CBP interacts with chromatin, alter patterns of CBP-dependent histone acetylation and change gene expression. Together, our work highlights how IDRs with different sequences, spatially segregated in the same protein, can cooperate to shape protein function.

molecular biology↗

bioRxiv2023

Notch signalling influences cell fate decisions and HOX gene induction in axial progenitors

The generation of the post-cranial embryonic body relies on the coordinated production of spinal cord neurectoderm and presomitic mesoderm cells from neuromesodermal progenitors (NMPs). This process is orchestrated by pro-neural and pro-mesodermal transcription factors that are co-expressed in NMPs together with Hox genes, which are critical for axial allocation of NMP derivatives. NMPs reside in a posterior growth region, which is marked by the expression of Wnt, FGF and Notch signalling components. While the importance of Wnt and FGF in influencing the induction and differentiation of NMPs is well established, the precise role of Notch remains unclear. Here, we show that the Wnt/FGF-driven induction of NMPs from human embryonic stem cells (hESCs) relies on Notch signalling. Using hESC-derived NMPs and chick embryo grafting, we demonstrate that Notch directs a pro-mesodermal character at the expense of neural fate. We show that Notch also contributes to activation of HOX gene expression in human NMPs, partly in a non cell-autonomous manner. Finally, we provide evidence that Notch exerts its effects via the establishment of a negative feedback loop with FGF signalling.

developmental biology↗