Biology subjects

Nowakowska, A.

Publications and source records attributed to Nowakowska, A..

4 recordsLinked to original sources

bioRxiv2026

Nanoscale insights into chromatin integrity molecular rearrangements upon DNA damage response

DNA Double Strand Breaks (DSBs) threaten genomic stability, leading to cell death, chromosomal rearrangements, and cancer-driving mutations. Therefore, the effective repair mechanisms are essential for maintaining genomic stability and ensuring cellular survival across diverse organisms. At the core of this process lies chromatin integrity, which facilitates the local DNA conformational changes, regulating accessibility to repair proteins and other biomolecules. To deepen our understanding of the DNA damage response pathway, the local molecular mechanisms regulating the interplay between DNA damage formation and alterations in chromatin conformation must be investigated at the nanoscale level. Here, we integrated atomic force microscope-infrared spectroscopy (AFM-IR) and confocal fluorescence microscopy to explore local chemical modifications in DNA structure and chromatin integrity in metaphase chromosomes and chromosomal aberrations isolated from cells treated with the chemotherapeutic agent, bleomycin. Our findings reveal changes in secondary protein structures, indicating the engagement of DNA repair proteins with high {beta}-sheet content. Nanospectroscopic mapping resolved the alterations in DNA condensation along the chromosomes. Moreover, we observed global DNA demethylation, particularly the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), correlating with increased DSBs. We conclude that these transitions in protein conformation and DNA methylation correlate with chromatin relaxation and enhanced accessibility for the repair protein.

biophysics↗

bioRxiv2025

Cell-in-Water: measurements using Optical Photothermal Infrared spectroscopy

Conventional Fourier transform infrared absorption spectroscopy (FT-IR) allows for measurements of spatial resolution comparable to a whole cell size, which are performed on dried cells to avoid water interference. However, such conditions can disrupt the native morphology and chemical composition of cells, whilst not providing for crucial sub-cellular biochemical detail. Pump-probe Optical Photothermal Infrared (O-PTIR) Microscopy offers high-resolution measurements of cells, but the challenge of measuring cells without disturbing their morphology remains. To overcome this, we developed a protocol enabling O-PTIR measurements of cells in aqueous environments using a silicone-sealed microchamber, which allows imaging of live cells in 2 {micro}l of buffer for up to 4 hours. Analysis of O-PTIR data obtained from hydrated and dehydrated cells confirms that hydrated conditions better preserve subcellular organization, as evidenced by secondary structure changes of proteins. We also propose a step-by-step algorithm that can improve image quality through ratiometric analysis, histogram assessment, and the application of RGB overlays. This approach allows for visualization of multiple subcellular components, such as the nucleus or lipid droplets. Trough the combination of "sandwich" sample configuration and O-PTIR, we offer a promising strategy to advance O-PTIR measurements of cells in water, which have long been considered a major challenge. A direct comparison of O-PTIR with Raman microscopy highlights the complementarity of these two techniques and similar spatial resolution.

biophysics↗

bioRxiv2025

Quantifying lipids in human cells using Raman spectroscopy

Withdrawal statementThe authors have withdrawn this manuscript because they have identified several inaccuracies that require further refinement. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.

biophysics↗

bioRxiv2022

Baculoviral COVID-19 Delta DNA vaccine cross-protects against SARS-CoV2 variants in K18-ACE2 transgenic mice

After severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) made the world tremble with a global pandemic, SARS-CoV2 vaccines were developed. However, due to the coronaviruss intrinsic nature, new variants emerged, such as Delta and Omicron, refractory to the vaccines derived using the original Wuhan strain. We developed an HERV-enveloped recombinant baculoviral DNA vaccine against SARS-CoV2 (AcHERV-COVID19S). A non-replicating recombinant baculovirus that delivers the SARS-CoV2 spike gene showed a protective effect against the homologous challenge in a K18-hACE2 Tg mice model; however, it offered only a 50% survival rate against the SARS-CoV2 Delta variant. Therefore, we further developed the AcHERV-COVID19 Delta vaccine (AcHERV-COVID19D). Cross-protection experiments revealed that mice vaccinated with the AcHERV-COVID19D showed 100% survival upon challenge with Delta and Omicron variants and 71.4% survival against prototype SARS-CoV2. These results support the potential of the viral vector vaccine, AcHERV-COVID19D, in preventing the spread of coronavirus variants such as Omicron and SARS-CoV2 variants. Author SummaryAfter the SARS-CoV2 pandemic, it is known that the existing vaccine has diminished efficacy against the emerging variants. We developed a baculoviral COVID19 DNA vaccine for the Delta variant (AcHERV-COVIS19D). Compared to AcHERV-COVID19S, designed to protect from the prototype of SARS-CoV2, AcHERV-COVID19D elicited higher humoral and cellular immunity and showed perfect protection against SARS-CoV2 delta strain and Omicron challenge. The broad and robust cellular immunity of the AcHERV-COVID19D vaccine appears to have played a significant role in the cross-protection of the Omicron variant. Our AcHERV-COVID19D can be a potential vaccine against emerging SARS-CoV2 variants.

immunology↗