Biology subjects

Rzyman, W.

Publications and source records attributed to Rzyman, W..

4 recordsLinked to original sources

bioRxiv2026

Correction of the cytosine deamination artifacts in FFPE-based sequencing experiments

Formalin-fixed, paraffin-embedded (FFPE) tissues remain an essential resource for molecular studies, yet formalin-induced cytosine deamination introduces characteristic C>T/G>A artifacts that compromise the accuracy of next-generation sequencing (NGS) analyses. Numerous computational methods and enzymatic DNA repair strategies have been proposed to reduce these artifacts, but no systematic comparison across tools and experimental conditions exists. Here, we evaluate the performance of seven computational approaches (SOBDetector, Ideafix, MicroSEC, FFPolish, DeepOmics FFPE/FFPE-PLUS, FFPErase) together with the NEBNext(R) FFPE DNA Repair Mix v2, a multi-enzyme repair system applied during DNA preparation. Using three independent datasets, one based on whole genome sequencing (CGCI-BL) and two on whole exome sequencing (TCGA-PC and SUT-LUAD, the latter containing enzymatically repaired samples), and matched fresh-frozen samples as the gold standard, we assess precision, sensitivity, and artifact reduction efficiency across all methods. We further examine the potential synergy between enzymatic repair and post-sequencing computational filtering. Our results provide practical guidelines for FFPE artifact correction and demonstrate that enzymatic treatment provides the best results, while among the computational methods, FFPErase offers the most robust reduction of cytosine deamination artifacts while maximizing the retention of true somatic variants. KEY MESSAGESO_LIFormalin fixation in FFPE samples introduces artifacts that can significantly affect the accuracy of NGS analyses. C_LIO_LIAmong the evaluated approaches, enzymatic repair using NEBNext(R) FFPE DNA Repair Mix v2 achieves the most effective reduction of sequencing artifacts. C_LIO_LIComputational methods vary in performance, with FFPErase showing the most robust balance between artifact removal and retention of true somatic variants. C_LIO_LICombining enzymatic repair with computational filtering did not lead to consistent improvements in performance across datasets. C_LI

bioinformatics↗

bioRxiv2026

Benchmarking five extracellular vesicle proteomics workflows, including low-input Exo-insert and Exo-SP3, for deep mass spectrometry profiling

Small extracellular vesicles (sEVs) are key mediators of intercellular communication, influencing diverse pathological processes, including cancer. While mass spectrometry (MS) has enabled the proteomic analysis of sEVs, sample preparation losses remain a critical bottleneck, particularly for scarce tissue-derived sEVs (Ti-EVs). Here, we systematically benchmark five proteomic workflows introducing Exo-insert, a novel single-vessel method, and Exo-SP3, across both Ti-Evs and cell culture-derived sEVs (CCM-EVs) at low input (0.5-4 {micro}g). Exo-insert and Exo-SP3 enable the identification of [~]1100 protein groups from as little as 0.5 {micro}g sEV input. Notably, optimal sample preparation for MS is source-dependent: Exo-insert and Exo-SP3 display divergent performance across sEV sources. Comparative DDA/DIA analyses establish sample preparation as the primary determinant of proteome recovery, offering a practical framework that matches workflows to sEV amounts and source-specific content for biomarker discovery.

cancer biology↗

bioRxiv2022

Major Histocompatibility Complex class I heavy chains localize in both cytoplasmic and nuclear compartment

The Major Histocompatibility Complex class I (MHC-I) molecules present antigenic peptides (AP) to CD8+ T cells for self versus non-self recognition. Loading of AP on MHC-I takes place in the endoplasmic reticulum (ER), upon shuttling of cytoplasmic AP substrates to the ER. Understanding of this process has been influenced by the view that MHC-I antigens are produced from the proteasomal degradation of cellular proteins. Recent observations on the intronic and untranslated region-derived peptides as well as on the non-AUG translation products presented on the MHC-I open the possibility that antigenic peptides can derive from pre-spliced mRNAs translated in the nuclear compartment. In this brief report, we show that a fraction of human MHC-I molecules (human leukocyte antigens type A, HLA-A) is present in the nuclei of cells, in the proximity of histone H2B. With this finding, we hope to initiate a new direction of research on the nuclear role of MHC-I and ask whether the loading of antigens can take place in the nuclear compartment.

immunology↗

bioRxiv2022

Keap1-resistant ΔN-Nrf2 isoform does not translocate to the nucleus upon electrophilic stress

The Nrf2 pathway is an essential defense pathway in a cell. It responds to oxidative and electrophilic stress via derepression of Nrf2 from Keap1-Cul3-mediated degradation, accumulation of Nrf2 in the nucleus and transcriptional activation of a number of detoxifying and cell protective Nrf2 target genes. Here we report that normal and cancer cells also express the N-terminally truncated Nrf2 isoform ({Delta}N-Nrf2), which originates from an alternative promoter. Co-immunoprecipitation together with molecular dynamics simulation showed that the binding between {Delta}N-Nrf2 and Keap1 is impaired, resulting in the much higher stability of this form. {Delta}N-Nrf2 is retained in the cytoplasm in response to electrophilic stress, indicating that it does not regulate transcription under the same stress stimuli as the full-length Nrf2. Altogether this data suggests that Nrf2 has other functions in cells than transcriptional activation of genes, which most probably rely on the protein-protein interactions in the cytoplasm. The regulation between these functions takes place on the level of transcription. Significance StatementThis work signifies the importance of alternative transcription in assigning the function to the produced protein. Nrf2 transcripts produced from the second promoter of the Nrf2 gene give rise to the N-terminally truncated Nrf2 form ({Delta}N-Nrf2), which is retained in the cytoplasm upon stress, thus it has a different role in cells than transcriptional regulation. {Delta}N-Nrf2 is resistant to the Keap1-Cul3 degradation pathway and is highly expressed in all tested cell types. This work points to the new, cytoplasmic role of Nrf2 in cells, determined at the level of transcription.

cell biology↗