Biology subjects

Sbardella, G.

Publications and source records attributed to Sbardella, G..

3 recordsLinked to original sources

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↗

bioRxiv2026

Regulation of BCL11A DNA binding and expression in human erythrocyte precursor HUDEP-2 cells

BCL11A is a transcription factor crucial for neurodevelopment and hematopoiesis. It regulates the developmental switch from fetal hemoglobin (HbF) to adult hemoglobin and is a major therapeutic target for sickle cell disease and {beta}-thalassemia. BCL11A exists in multiple isoforms, including the L isoform (containing a single two-finger ZF2-3 DNA-binding domain) and the XL isoform (containing two arrays: the two-finger ZF2-3 and the three-finger ZF4-6). We used three approaches to investigate BCL11A functions. First, we examined DNA recognition by BCL11A, which preferentially binds the short 6-bp DNA motif TGNCCA. ZF4-6 recognizes all four variants of this motif with distinct strand-specific interactions: TGTCCA on the top strand, TG(A/C)CCA on the complementary strand, and the palindromic TGGCCA on either strand. ZF2-3 also binds TGTCCA from the top strand, featuring a unique thymine interaction by ZF2 Phe388. Motif multiplicity within BCL11A binding sites may promote BCL11A oligomerization by enabling multiple ZF arrays to engage DNA simultaneously. Second, we treated HUDEP-2 cells (which express adult hemoglobin) with inhibitors targeting three epigenetic silencing marks - DNA methylation, histone H3 lysine 9 methylation or H3 lysine 27 methylation. All treatments, individually or in combination, increased HbF expression to varying degrees. Notably, FTX6058 markedly reduced BCL11A transcription and translation (likely via effects on LIN28B), while EML741 caused a partial reduction. Third, we screened 213 pomalidomide- and lenalidomide-derived compounds and quantified proportions of HbF+ cells by flow cytometry. Effects of four compounds were analyzed by protein mass spectrometry. Although BCL11A levels themselves were unchanged, all four compounds selectively decreased levels of known pomalidomide targets, with consistently decreased levels of the zinc-finger proteins IKZF1 and ZFP91. Together, our studies clarify how BCL11A recognizes DNA, how its expression can be modulated epigenetically, and how small-molecule degraders influence its regulatory network, providing new avenues for HbF reactivation therapies.

biochemistry↗

bioRxiv2025

Reverse transcriptase inhibitors induce autophagy in a LINE-1 ORF1p-dependent manner

Human Long Interspersed Nuclear Element-1 (LINE-1) retrotransposons propagate throughout the genome via reverse-transcribed RNA intermediates. LINE-1 expression is pervasive in cancer. Functional LINE-1s encode two proteins: ORF1p, an RNA-binding protein, and ORF2p, harboring reverse transcriptase and endonuclease activities. Reverse transcriptase inhibitors, including non-nucleoside (NNRTI) and nucleoside (NRTI) inhibitors, inhibit cancer cell proliferation and antagonize cancer progression. We previously found that two NNRTIs induced DNA damage, nuclear lamin rupture, micronuclei formation, and autophagy in prostate cancer cells. We now find that two different RTIs up-regulate LINE-1 mRNA expression and ORF1p abundance in nuclei, triggering ORF1p interactions with lamin B1 and with DNA damage factors. ORF1p accumulates within micronuclei with damaged DNA and with the autophagy receptor p62. We further demonstrate that inhibiting autophagy, or decreasing ORF1p levels, prevent DNA damage and preserve lamin B1 integrity, uncoverig a role of LINE-1-ORF1p in the autophagy response of cancer cells, independent on retrotranscription events.

cell biology↗