Biology subjects

Wisniewski, J.

Publications and source records attributed to Wisniewski, J..

14 recordsLinked to original sources

bioRxiv2026

Identification of different sequence properties between HIV-1 DNA and RNA across subtypes using the k-mer-based approach

Advanced analytical tools that enable mining of the masked features hidden in intricate datasets and strengthening the biological interpretation of multigenomic outputs hold paramount importance. At present, HIV-1 subtyping remains a challenging task in a great part due to analytical tool discordance. To tackle this issue, in this study, we present an updated version of a k-mer-based approach, PORT-EK-v2, a streamlined bioinformatic pipeline, allowing for a comparison of multiple genomic datasets and identification of over-represented genomic regions, k-mers, related to specific origins of datasets. Using PORT-EK-v2, we exemplified that intrinsic sequence patterns between HIV-1 DNA and RNA are distinct across group M HIV-1 subtypes. Furthermore, we showcased that "isolate k-mer count", a predictive variable computed in this work, could serve as a default choice in classifying the HIV-1 DNA versus RNA sequences across subtypes. Lastly, results based on network-based analyses and Markov chain Monte Carlo modeling unveiled a clear discontinuation of a random walk throughout the network properties corresponding to each tested group of HIV-1 subtypes, confirming the specificity of enriched k-mer retrieved by PORT-EK-v2 and the genomic diversity across group M HIV-1 subtypes. Source code for PORT-EK-v2 is at https://github.com/Quantitative-Virology-Research-Group/PORT-EK-version-2 and is freely available.

bioinformatics↗

bioRxiv2025

A Robust Crystallographic Platform for High-Throughput β-Catenin Ligand Discovery

This study presents a robust crystallographic platform for assessing compounds binding to {beta}-catenin. We developed a standardized protein production protocol for the armadillo domain of {beta}-catenin (BC-ARM) and performed biophysical screens using Surface Plasmon Resonance (SPR) and Differential Scanning Fluorimetry (DSF). These findings led to the successful determination of the co-crystal structure of BC-ARM with compound 1 binding to previously reported site but distinct from known transcription factor binding sites. To broaden the search for novel BC binding sites, we utilized FragLites library with a cyclic peptide-stabilized BC-ARM construct. This yielded two high-resolution co-crystal structures identifying a previously unreported binding hotspot. Recognizing the limitations of the cyclic peptide-bound construct for general screening, we designed a novel, truncated BC-ARM construct. This new construct eliminates unstructured regions, reliably producing high-quality, diffracting crystals suitable for high-throughput crystallographic studies. In conclusion, the ligand-bound {beta}-catenin structures and this novel, robust BC-ARM construct establish a powerful platform for further {beta}-catenin investigation.

molecular biology↗

bioRxiv2025

Exploration of chemical probes and conformational flexibility of GID4 - the substrate receptor of human CTLH E3 ligase complex

The application of targeted protein degradation (TPD) is currently constrained by the limited availability of low-molecular-weight molecules that can recruit E3 ligases other than CRBN (Cereblon) or VHL (Von Hippel-Lindau ligase). In this study, we present the structure-based drug design (SBDD) of high-affinity ligands that engage E3 ligase GID4 (Glucose-induced degradation protein 4) in biophysical and cellular experiments. Through structural studies and molecular modeling, we identified three clusters of compounds that induce distinct conformations of GID4. We characterized potential exit vectors and used the most promising ligand as a building block to prepare bifunctional degraders in the form of proteolysis-targeting chimeras (PROTACs). Although ternary complex formation was successful in vitro, degradation of BRD4 was not observed, highlighting the need for further optimization of the degraders. Finally, we theoretically investigated the likelihood of the identified GID4 conformations participating in protein-protein interactions mediated by molecular glue mechanisms. We believe the expanded ligand diversity discovered in this study may pave the way for tuning the selectivity and efficacy of interactions involving GID4 and its neosubstrates. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/662521v3_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@19187e8org.highwire.dtl.DTLVardef@171abd1org.highwire.dtl.DTLVardef@1c75a5forg.highwire.dtl.DTLVardef@fe17fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Targeted degradation of GSPT1 and NEK7 by a molecular glue prodrug for treatment of HCC

Targeted Protein Degradation (TPD) technology, in the form of CRBN-modulating molecular glues, offers numerous unprecedented therapeutic benefits as evidenced by the success of approved high-value immunomodulatory imide drugs (IMiDs) such as lenalidomide and pomalidomide. Building upon these successes, we employed a small CRBN-focused library of molecular glues in a phenotypic screen against hepatocellular carcinoma (HCC) cell lines. While the original library was primarily designed to target SALL4, we identified additional CRBN substrates, including GSPT1, NEK7, and CK1, whose degradation potently induced cell death in HCC cell lines. Subsequent lead optimization efforts yielded a compound, ABS-752, which demonstrated superior in vitro and in vivo activity through the potent degradation of GSPT1. Notably, ABS-752 does not form ternary complexes with CRBN and the neosubstrates. Further investigations revealed that ABS-752 is a prodrug activated by the monoamine oxidase, VAP-1, to an aldehyde intermediate and subsequently to the active molecule, ABT-002. VAP-1, which is overexpressed in cirrhotic liver, was identified as the primary monoamine oxidase responsible for the conversion of ABS-752. ABS-752 is currently in clinical trials for the treatment of HCC.

molecular biology↗

bioRxiv2025

The influence of HIV sense and antisense transcripts on stochastic HIV gene expression and reactivation

In this study, we established dozens of single provirus-infected cellular clones offering various transcriptional phenotypes of HIV. We proposed that stochastic fluctuations in HIV transcription can appear at, at least, two levels: (1) the chromosomal landscape and (2) the in situ HIV integration site. In the former case, proviruses integrating at different genomic locations demonstrated a variety of transcriptional bursting and can be classified in noise space constructed based on the parameters associated with the coefficient of variation and using a mathematical model fitting a curve of exponential decay. In the latter case, stochastic HIV transcription can be unveiled through its phenotypic bifurcation and tended to be a pure epigenetic phenomenon: the identical provirus demonstrated fluctuations in its transcription with an elevated frequency. We observed similar expression patterns between sense and antisense RNA transcripts. Notably, both HIV long terminal repeats reacted to drug stimulation and may reveal distinct behaviors. Overall, our data suggest that HIV antisense transcripts could be involved in the stochastic nature of HIV transcription.

systems biology↗

bioRxiv2025

Mechanistic insights and clinical implications of cross-reactive anti-prophage antibodies and bacterial heteroresistance on phage therapeutic failure

Phage therapy is an exciting strategy against antimicrobial-resistant bacterial infections, but critical knowledge gaps regarding its clinical application persist. Studying a patient with a life-threatening, chronic bacterial infection who failed phage therapy, we uncovered important biological concepts with direct translational impact. Using longitudinal clinical samples, we found that patients can harbour pre-existing antibodies against active prophages induced from the genome of the causative pathogen. Notably, these antibodies can contribute to clinical failure by cross-reacting with and effectively neutralising therapeutic phage. We also uncovered bacterial heteroresistance, characterised by bacterial subpopulations from the initial infection with reduced phage susceptibility, as a further contributor to treatment failure. These findings highlight the intricate interplay between host immunology, bacterial genetic diversity and phage biology, bearing broad significance for clinical phage therapy. Future phage therapy patients, especially those with chronic infections, should be screened for antiphage immunity and bacterial heteroresistance prior to phage treatment.

microbiology↗

bioRxiv2024

LC-MS/MS characterization of SOBERANA(R)02, a receptor binding domain-tetanus toxoid conjugate vaccine against SARS-CoV-2

SOBERANA(R)02 is a safe and effective conjugate vaccine against SARS-CoV-2, produced using the maleimide-thiol chemistry. In this vaccine, the Cys538 in the recombinant receptor binding domain (RBD) of SARS-CoV-2 is linked, through a thiosuccinimide linker, to lysine residues of tetanus toxoid (TT) preparation. LC-MS/MS analysis revealed that TT is a complex mixture of proteins, similar to other TTs where the detoxified tetanus neurotoxin (d-TeNT) has been shown to be the most abundant protein (30-56%), regardless the quantification method used. The fifteen most abundant proteins account for approximately 78% of the total proteins. LC-MS/MS analysis of the activation process showed that 102 out of 107 lysine residues in the d-TeNT incorporated a maleimide group. In contrast, when tryptic peptides isolated by Ni2+-NTA affinity chromatography, were analyzed by LC-MS/MS, only 22 Lys residues in the d-TeNT were cross-linked to the RBD C-terminal tryptic peptide (538CVNF541-HHHHHH), probably due to steric hindrance. Twelve and eighteen conjugation sites were assigned based on the identification of linear peptides carrying a conjugated lysine residues ({Delta}m = +1454.58 Da or {Delta}m = +1472.59 Da) and cross-linked peptides with stabilized linker forms, respectively. Eight conjugation sites were coincidently assigned by both strategies. The assignment of the conjugation sites was manually validated by observed regularities (z[≥]3+, XIC, immonium ions, specific linker fragment ions) not considered by the identification software (PEAKS, Kojak and pLink2). The RBD was also conjugated, but to a lesser extent, to ten other low abundance carrier proteins. To our knowledge, this work is the first report of conjugation site assignment in a TT-based conjugate vaccine.

biochemistry↗

bioRxiv2024

Identification of potential SARS-CoV-2 genetic markers resulting from host domestication.

We developed a k-mer-based pipeline, namely the Pathogen Origin Recognition Tool using Enriched K-mers (PORT-EK) to identify genomic regions enriched in the respective hosts after the comparison of metagenomes of isolates between two host species. Using it we identified thousands of k-mers enriched in US white-tailed deer and betacoronaviruses in bat reservoirs while comparing them with human isolates. We demonstrated different coverage landscapes of k-mers enriched in deer and bats and unraveled 148 mutations in enriched k-mers yielded from the comparison of viral metagenomes between bat and human isolates. We observed that the third position within a genetic codon is prone to mutations, resulting in a high frequency of synonymous mutations of amino acids harboring the same physicochemical properties as unaltered amino acids. Finally, we classified and predicted the likelihood of host species based on the enriched k-mer counts. Altogether, PORT-EK showcased its feasibility for identifying enriched viral genomic regions, illuminating the different intrinsic tropisms of coronavirus after host domestication. TeaserA measure of enriched viral genomic correlates resulting from host domestication as a potential predictor of zoonotic risk.

systems biology↗

bioRxiv2024

Distinguishable topological properties of functional genome networks in HIV-1 reservoirs

HIV-1 reservoirs display heterogeneous nature, lodging both intact and defective proviruses. Recent evidence has shed light on their difference, particularly in the context of immune-mediated selection. To deepen our understanding of such heterogeneous HIV-1 reservoirs and their functional implications, we pioneered the integration of basic concepts of graph theory to characterize the composition of HIV-1 reservoirs. Our analysis revealed noticeable topological properties in networks, featuring immunologic signatures enriched by genes harboring intact and defective proviruses, when comparing antiretroviral therapy (ART)-treated HIV-1-infected individuals and elite controllers. The key variable, the rich factor, played a pivotal role in classifying distinct topological properties in networks. The host gene expression strengthened the accuracy of classification between elite controllers and ART-treated patients. Overall, our work provides a prime example of leveraging genomic approaches alongside mathematical tools to unravel the complexities of HIV-1 reservoirs.

genomics↗

bioRxiv2023

BRD4 directs mitotic cell division by inhibiting DNA damage

BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth.

cell biology↗

bioRxiv2021

Kinetic principles underlying pioneer function of GAGA transcription factor in live cells

How pioneer factors interface with chromatin to promote accessibility for transcription control is poorly understood in vivo. Here, we directly visualize chromatin association by the prototypical GAGA pioneer factor (GAF) in live Drosophila hemocytes. Single-particle tracking reveals that the majority of GAF is chromatin-bound, with a stable-binding fraction showing nucleosome-like confinement residing on chromatin for over 2 minutes, far longer than the dynamic range of most transcription factors. These kinetic properties require the full complement of GAFs DNA-binding, multimerization and intrinsically disordered domains, and are autonomous from recruited chromatin remodelers NURF and PBAP, whose activities primarily benefit GAFs neighbors such as HSF. Evaluation of GAF kinetics together with its endogenous abundance indicates that despite on-off dynamics, GAF constitutively and fully occupies chromatin targets, thereby providing a temporal mechanism that sustains open chromatin for transcriptional responses to homeostatic, environmental, and developmental signals.

molecular biology↗

bioRxiv2020

Spatio-Temporal Coordination Of Transcription Preinitiation Complex Assembly In Live Cells

Transcription initiation by RNA polymerase II (Pol II) requires preinitiation complex (PIC) assembly at gene promoters. In the dynamic nucleus where thousands of promoters are broadly distributed in chromatin, it is unclear how ten individual components converge on any target to establish the PIC. Here, we use live-cell, single-molecule tracking in S. cerevisiae to document subdiffusive, constrained exploration of the nucleoplasm by PIC components and Mediators key functions in guiding this process. On chromatin, TBP, Mediator, and Pol II instruct assembly of a short-lived PIC, which occurs infrequently but efficiently at an average promoter where initiation-coupled disassembly may occur within a few seconds. Moreover, PIC exclusion by nucleosome encroachment underscores regulated promoter accessibility by chromatin remodeling. Thus, coordinated nuclear exploration and recruitment to accessible targets underlies dynamic PIC establishment in yeast. Collectively, our study provides a global spatio-temporal model for transcription initiation in live cells.

molecular biology↗

bioRxiv2020

Dual-view light-sheet imaging through tilted glass interface using a deformable mirror

Light-sheet microscopy has become one of the primary tools for imaging live developing organisms because of its high speed, low phototoxicity, and optical sectioning capabilities. Detection from multiple sides (multi-view imaging) additionally allows nearly isotropic resolution via computational merging of the views. However, conventional light-sheet microscopes require that the sample is suspended in a gel to allow optical access from two or more sides. At the same time, the use of microfluidic devices is highly desirable for many experiments, but geometric constrains and strong optical aberrations caused by the coverslip titled relative to objectives make the use of multi-view lightsheet challenging for microfluidics. In this paper we describe the use of adaptive optics (AO) to enable multi-view light-sheet microscopy in such microfluidic setup by correcting optical aberrations introduced by the tilted coverslip. The optimal shape of deformable mirror is computed by an iterative stochastic gradient-descent algorithm that optimizes PSF in two orthogonal planes simultaneously. Simultaneous AO correction in two optical arms is achieved via a knife-edge mirror that splits excitation path and combines the detection path. We characterize the performance of this novel microscope setup and, by dual-view light-sheet imaging of C.elegans inside a microfluidic channel, demonstrate a drastic improvement of image quality due to AO and dual-view reconstruction. Our microscope design allows multi-view light-sheet microscopy with microfluidic devices for precisely controlled experimental conditions and high-content screening.

bioengineering↗

bioRxiv2020

Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription of noncoding RNAs prior to premature termination, in addition to transcription of mRNAs, are responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

molecular biology↗