Biology subjects

Milanova, V.

Publications and source records attributed to Milanova, V..

3 recordsLinked to original sources

bioRxiv2026

DePARylation prevents DNA replication-driven PARP1 condensation

PARG, the primary enzyme responsible for the reversal of PARP1-mediated poly(ADP-ribosyl)ation, has attracted considerable attention as a therapeutic target in cancer. Yet, the mechanisms underlying PARG inhibitor (PARGi) efficacy remain elusive. Herein, we demonstrate that PARGi prolongs PARP1 residence at damaged chromatin in a manner mechanistically distinct from PARP-inhibitor-induced trapping. That is, PARGi triggers the formation of PAR-driven, FUS-enriched PARP1 nuclear condensates upon DNA damage. Importantly, unrestrained S-phase PARylation during Okazaki fragment maturation also elicited PARP1 condensation in a manner directly reflecting intrinsic PARGi sensitivity, with FEN1 co-inhibition enhancing both PARP1 condensate formation and cytotoxicity. Finally, we discover that dePARylation prevents the rapid nuclear extrusion of PARP1 upon S-phase entry, a phenomenon that is reversible and could undermine PARP1-dependent nuclear processes. Together, our findings reveal that dePARylation precludes the replication-driven condensation of PARP1 and identify Okazaki fragment maturation as a targetable vulnerability that exacerbates condensation and PARGi cytotoxicity.

molecular biology↗

bioRxiv2022

Preclinical assessment of Fibroblast Activation Protein-specific MRI as an alternative to PSMA for image-guided focal therapy of prostate cancer

Accurate and precise delineation of gross tumour volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumours, such as prostate cancer. Magnetic resonance imaging of tumour molecular targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-LINAC. Fibroblast activation protein (FAP) is a transmembrane protein overexpressed in stromal components of >90% of epithelial carcinomas. Herein we compare targeted MRI of gold standard PSMA with FAP in the delineation of orthotopic tumours in a mouse model of prostate cancer. Control (no ligand), FAP and PSMA-targeting iron oxide nanoparticles were prepared with modification of an MRI agent (FerroTrace). Mice with orthotopic LNCaP tumours underwent T2-weighted 3D MRI 24 hours after intravenous injection of contrast agents. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles, which was most pronounced on the tumour periphery. FAP-targeted MRI increased the proportion of tumour contrast enhancing black pixels by 13.37% when compared to PSMA. Furthermore, analysis of changes in R2 values between healthy prostates and LNCaP tumours indicated an increase in contrast enhancing pixels in the tumour border of 15%, when targeting FAP, in contrast to PSMA This study demonstrates preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.

bioengineering↗

bioRxiv2022

Magnetic Enrichment of Immuno-Specific Extracellular Vesicles for Mass Spectrometry Using Biofilm-Derived Iron Oxide Nanowires

Immuno-specific enrichment of extracellular vesicles (EVs) can provide important information into cellular pathways underpinning various pathologies and for non-invasive diagnostics, including mass spectrometry-based analyses. Herein, we report an optimised protocol for immuno-magnetic enrichment of specific EV subtypes and their subsequent processing with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, we conjugated placental alkaline phosphatase (PLAP) antibodies to magnetic iron oxide nanowires (NWs) derived from bacterial biofilms and demonstrated the utility of this approach by enriching placental specific EVs (containing PLAP) from cell culture media. We demonstrate efficient PLAP+ve EV enrichment for both NW-PLAP and Dynabeads-PLAP, with high PLAP protein recovery (83.7{+/-}8.9% and 83.2{+/-}5.9%, respectively), high particle-to-protein ratio (7.5{+/-}0.7x109 and 7.1{+/-}1.2x109, respectively), and low non-specific binding of non-target EVs (7{+/-}3.2% and 5.4{+/-}2.2%, respectively). Furthermore, our optimized EV enrichment and processing approach identified 2518 and 2545 protein groups with LC-MS/MS for NW-PLAP and Dynabead-PLAP, respectively, with excellent reproducibility (Pearson correlation 0.986 and 0.988). These findings demonstrate that naturally occurring iron oxide NWs have comparable performance to current gold standard immune-magnetic beads. The optimized immunospecific EV enrichment for LC-MS/MS method provides a low-cost and highly-scalable yet efficient, high-throughput approach for quality EV proteomic studies.

bioengineering↗