Biology subjects

Rames, M. J.

Publications and source records attributed to Rames, M. J..

3 recordsLinked to original sources

bioRxiv2025

Automated Machine Learning Profiling with MAP-HR for Quantifying Homologous Recombination Foci in Patient Samples

Accurate visualization and quantification of homologous recombination (HR)-associated foci in readily available patient samples are critical for identifying patients with HR deficiency (HRD) when they present for care to guide polyADP ribose polymerase (PARP) inhibitors (PARPi) or platinum-based therapies. Immunofluorescence (IF) assays have the potential to accurately visualize DNA repair processes as punctate foci within the nucleus. To ensure precise HRD assessment, we developed MAP-HR, (Machine-learning Assisted Profiling of Homologous Recombination), a scalable machine-learning (ML) analysis platform to enable effective patient triage and therapeutic decision-making. This workflow integrates high-resolution four-channel IF imaging and automated analysis of Geminin (cell cycle states), RAD51 (HR repair),{gamma} H2AX foci (double strand breaks) and DAPI (nuclear localization) in both cultured cell lines and in a single formalin-fixed, paraffin-embedded (FFPE) patient sample. Using a spinning disk confocal microscope, we optimized imaging parameters to improve resolution and signal-to-noise ratio. Our MAP-HR pipeline uses nested nuclei and segmentation of foci to analyze the HR status of each cell, unlike competing bulk or single-foci marker assays, allowing evaluation of HR functional heterogeneity across and within patient biopsies. This approach facilitates robust comparisons of HR and foci-based processes across diverse cell populations and patient tissues, enabling scalable, translational research. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/639329v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@154d43eorg.highwire.dtl.DTLVardef@3a6fa6org.highwire.dtl.DTLVardef@782daborg.highwire.dtl.DTLVardef@1fe6c28_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2022

Selective enrichment of plasma cell-free messenger RNA in cancer-associated extracellular vesicles

Extracellular vesicles (EVs) have been shown as key mediators of extracellular small RNA transport. However, carriers of cell-free messenger RNA (cf-mRNA) in human biofluid and their association with cancer remain poorly understood. Here, we performed a transcriptomic analysis of size-fractionated plasma from lung cancer, liver cancer, multiple myeloma, and healthy donors. Morphology and size distribution analysis showed the successful separation of medium and small EVs and non-vesicular carriers. We developed a strategy to purify and sequence ultra-low amounts of cf-mRNA from vesicular and non-vesicular subpopulations with the implementation of RNA spike-ins to control for technical variability and to normalize for intrinsic drastic differences in the amount of cf-mRNA carried in each plasma fraction. We found that the majority of cf-mRNA was enriched and protected in EVs with remarkable stability in RNase-rich environments. We observed specific enrichment patterns of cancer-associated cf-mRNA in each vesicular and non-vesicular subpopulation. The EV-enriched differentiating genes were associated with specific biological pathways, such as immune systems, liver function, and toxic substance regulation in lung cancer, liver cancer, and multiple myeloma, respectively. Our results suggest that dissecting the complexity of EVs subpopulations illuminates their biological significance and offers a promising liquid biopsy approach.

genomics↗

bioRxiv2022

Multiplexed and millimeter-scale superresolution imaging of cells and tissue sections via prism-illumination and microfluidics-enhanced DNA-PAINT

Fluorescence nanoscopy has become increasingly powerful for biomedical research, but it has historically afforded a small field-of-view (FOV) around 50 {micro}m x 50 {micro}m at once and more recently up to ~200 {micro}m x 200 {micro}m. Efforts to further increase the FOV in fluorescence nanoscopy have thus far relied on the use of fabricated waveguide substrates, adding cost and sample constraints on the applications. Here we report PRism-Illumination and Microfluidics-Enhanced DNA-PAINT (PRIME-PAINT) for multiplexed fluorescence nanoscopy across millimeter-scale FOVs. Built upon the well-established prism-type total internal reflection microscopy, PRIME-PAINT achieves robust single-molecule localization with up to ~520 {micro}m x 520 {micro}m single FOVs and 25-40 nm lateral resolutions. Through stitching, nanoscopic imaging over mm2 sample areas can be completed in as little as 40 minutes per target. An on-stage microfluidics chamber facilitates probe exchange for multiplexing and enhances image quality particularly for formalin-fixed paraffin-embedded (FFPE) tissue sections. We demonstrate the utility of PRIME-PAINT by analyzing ~106 caveolae structures in ~1,000 cells and imaging entire pancreatic cancer lesions from patient tissue biopsies. By imaging from nanometers to millimeters with multiplexity and broad sample compatibility, PRIME-PAINT will be useful for building multiscale, Google-Earth-like views of biological systems.

biophysics↗