Biology subjects

Vega, S.

Publications and source records attributed to Vega, S..

3 recordsLinked to original sources

bioRxiv2026

Live immunogenic melanoma cells generated by etoposide treatment upregulate B2M and lose tumor-forming potential

Cytotoxic chemotherapy has been the primary cancer treatment modality for decades, however, chemotherapy drugs do not selectively target cancer cells. These drugs are toxic to all proliferating cells including immune cells that constitute the host-defense system against pathogens and cancer cells. In an effort to develop non-toxic combinations of chemotherapy and immunotherapies for cancer, we previously reported in Sriram et al. 2021 that etoposide-treated B16 melanoma cells expressing ovalbumin, when co-cultured in vitro with dendritic cells and T-cells, enhance T-cell activation. The live fraction of melanoma cells after etoposide treatment was critical to promote T-cell activation as dead cells alone or in combination with cell-free supernatants were not effective. In this study, we show that the live fraction of etoposide-treated B16-Ova cells, which gain the ability to stimulate T-cells, are growth-arrested, but remain viable past the co-incubation period with dendritic cells and T-cells post etoposide washout. Furthermore, using a soft-agar colony-formation assay as a proxy for tumor-forming potential, we show that these live etoposide-treated B16-Ova cells have drastically reduced ability to form colonies displaying a near-complete loss of anchorage-independent growth. We also show that etoposide treatment upregulates the production of Beta-2-microglobulin (B2M), a subunit of Class I MHC. Intriguingly, we observed using immunofluorescence that the live etoposide-treated B16-Ova cells show enhanced intracellular perinuclear B2M accumulation but B2M surface staining, while detectable, is not enhanced by etoposide treatment. Furthermore, while the live etoposide-treated B16-Ova cells express Class I MHC protein, there is only a marginal drug treatment-induced increase observed in the expression levels by western blotting. Our results suggest that T-cell activation by live etoposide-treated B16-Ova cells is likely not driven by drug treatment-induced enhancement in cell surface levels of Class I MHC and point to other mechanisms.

cancer biology↗

bioRxiv2026

Interlaboratory benchmarking of differential scanning fluorimetry reveals sources of variability in protein thermal stability parameters

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two phase interlaboratory benchmark using a shared VHH sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant reported apparent melting temperatures (Tm) showed good within instrument repeatability but moderate inter instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data acceptance criteria in addition to instrument dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent Tm depended strongly on the fluorescence readout. For this VHH, single wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF derived apparent unfolding enthalpies were more variable than apparent Tm. Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data quality criteria, and well characterised reference materials to monitor instrument dependent offsets.

biophysics↗

bioRxiv2019

In-depth and 3-Dimensional Exploration of the Budding Yeast Phosphoproteome

Phosphorylation is one of the most dynamic and widespread post-translational modifications regulating virtually every aspect of eukaryotic cell biology. Here we present a comprehensive phosphoproteomic dataset for budding yeast, comprised of over 30,000 high confidence phosphorylation sites identified by mass spectrometry. This single dataset nearly doubles the size of the known phosphoproteome in budding yeast and defines a set of cell cycle-regulated phosphorylation events. With the goal of enhancing the identification of functional phosphorylation events, we performed computational positioning of phosphorylation sites on available 3D protein structures and systematically identified events predicted to regulate protein complex architecture. Results reveal a large number of phosphorylation sites mapping to or near protein interaction interfaces, many of which result in steric or electrostatic \"clashes\" predicted to disrupt the interaction. Phosphorylation site mutants experimentally validate our predictions and support a role for phosphorylation in negatively regulating protein-protein interactions. With the advancement of Cryo-EM and the increasing number of available structures, our approach should help drive the functional and spatial exploration of the phosphoproteome.

biochemistry↗