Biology subjects

Daly, S.

Publications and source records attributed to Daly, S..

11 recordsLinked to original sources

bioRxiv2026

Defining the RNA Modification Landscape of Multiple Myeloma Reveals METTL3-Dependent m6A Regulation of NEAT1

RNA modifications play critical roles in gene regulation. N6-methyladenosine (m6A) is the most abundant modification on mRNA and long noncoding RNA (lncRNA) and regulates RNA processing, stability, and translation. RNA modifications are not well characterized in multiple myeloma (MM), a plasma cell malignancy characterized by relapse and disease progression, and the contribution of m6A-modified lncRNAs to the disease remains unclear. Here, we define the RNA modification landscape of MM by combining mass spectrometry, Nanopore Direct RNA sequencing, and methylated RNA immunoprecipitation sequencing. We identify 20 RNA modification types and > 15,000 m6A sites, including sites on 2,398 lncRNAs. Among these, we validate m6A sites on the paraspeckle-associated lncRNA NEAT1. Functional studies reveal that NEAT1 expression is regulated by the methyltransferase METTL3 and site-specific demethylation of a NEAT1 m6A site reduces MM cell viability. Single-cell RNA sequencing shows consistent NEAT1 enrichment in malignant plasma cells but minimal expression in healthy cells. These findings identify m6A-modified lncRNAs as key regulators of MM biology and establish NEAT1 as an epitranscriptomically controlled driver of MM cell survival.

cancer biology↗

bioRxiv2025

Protein-protein interactions drive differences in the spatiotemporal dynamics of transcription factors NANOG and SOX2 in naïve pluripotent cells

Maintenance of naive pluripotency requires core transcription factors (TFs) like SOX2 and auxiliary TFs like NANOG, yet molecular mechanisms governing their intra-nuclear dynamics and DNA binding interactions remain unclear. Here, using high-density 3D single-molecule light-field microscopy combined with novel spatiotemporal analysis pipelines, we track SOX2 and NANOG dynamics in live cells. Despite lower protein abundance, NANOG displays a similar chromatin-bound fraction to SOX2. This arises partially because, while both TFs undergo frequent transient non-specific binding interactions ([~]0.5-0.7s), NANOG exhibits more stable specific binding ([~]25s vs [~]16s). Both TFs also assemble into phase-separated domains of [~]400 nm containing both freely diffusing and chromatin-bound proteins, which further influences their dynamics. Strikingly, NANOGs protein-protein interaction domain markedly increases chromatin residence time (>5-fold) and the size of these phase-separated domains. Our work uncovers how NANOG and SOX2 stabilise gene regulatory networks that maintain naive pluripotency while providing quantitative pipelines for dissecting spatiotemporal TF dynamics.

cell biology↗

bioRxiv2025

SMOLM-LFM: ratiometric single molecule orientation without polarizers

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the methods potential and practical limitations.

bioengineering↗

bioRxiv2025

Volumetric single-molecule tracking inside subcellular structures

The molecular interactions that underpin all cellular functions depend on molecular motion within three-dimensional environments. Large depth-of-field single-molecule localization microscopy (3D-SMLM) methods facilitate these measurements, but their increased optical complexity and bespoke post-processing pipelines can sacrifice important cellular context. Here we combine single-molecule light-field microscopy (SMLFM) with widefield Fourier light-field microscopy for correlative volumetric organelle imaging. The instantaneous acquisition of subcellular volumes improves the sensitivity of molecular organization and diffusion measurements through the rejection of non-specific localizations. We first demonstrate our approach by measuring the molecular organization of a nuclear-localized HaloTag protein relative to cell nuclei. Next, we characterize the molecular diffusion of the soluble protein, calreticulin, in the context of 1-antitrypsin deficiency, which revealed an increase in heterogeneous motion within endoplasmic reticulum inclusions.

biophysics↗

bioRxiv2024

3D super-resolution imaging of PSD95 reveals an abundance of diffuse protein supercomplexes in the mouse brain

PSD95 is an abundant scaffolding protein that assembles multiprotein complexes controlling synaptic physiology and behavior. Confocal microscopy has previously shown that PSD95 is enriched in the postsynaptic terminals of excitatory synapses and 2D super-resolution microscopy further revealed that it forms nanoclusters. In this study, we utilized 3D super-resolution microscopy to examine the nanoarchitecture of PSD95 in the mouse brain, characterizing over 8 million molecules. While we were able to identify structural subtypes previously reported, imaging in 3D allowed us to classify these with higher accuracy. Furthermore, 3D super-resolution microscopy enabled the quantification of protein levels, revealing an abundance of PSD95 molecules existed outside of synapses as a diffuse population of supercomplexes, containing multiple copies of PSD95. Further analysis of the supercomplexes containing two units identified two populations: one that had PSD95 molecules separated by 39 {+/-} 2 nm, and a second with a separation of 94 {+/-} 27 nm. These results suggest that PSD95 supercomplexes containing multiple protein copies assemble outside the synapse and then integrate into the synapse to form a supramolecular nanocluster architecture.

biophysics↗

bioRxiv2024

Vortex light field microscopy: 3D spectral single-molecule imaging with a twist

We introduce vortex light field microscopy (VLFM), a novel method for snapshot 3D spectral single-molecule localization microscopy. Inspired by the azimuthal phase profile of optical vortices, we place an azimuthally oriented prism array immediately after the microlens array in a Fourier light field microscope (FLFM). This innovative arrangement causes the axial position and spectral peak for a point emitter to be encoded in the radial and azimuthal displacement of point-spread-function (PSF) respectively. This enables simultaneous detection of 3D position and emission peak of individual fluorophores with 25 nm spatial precision and 3 nm spectral precision over a 4 m depth of field (DOF). We illustrate the spectral scalability of our method by performing four-color 3D single particle tracking of freely diffusing fluorescent beads, and two-color 3D dSTORM imaging of microtubules and mitochondria in fixed COS-7 cells, without the need for spectrally distinct fluorophores.

biophysics↗

bioRxiv2024

LINC01432 binds to CELF2 in newly diagnosed multiple myeloma promoting short progression-free survival to standard therapy

Multiple Myeloma (MM) is a highly prevalent and incurable form of cancer that arises from malignant plasma cells, with over 35,000 new cases diagnosed annually in the United States. While there are a growing number of approved therapies, MM remains incurable and nearly all patients will relapse and exhaust all available treatment options. Mechanisms for disease progression are unclear and in particular, little is known regarding the role of long non-coding RNAs (lncRNA) in mediating disease progression and response to treatment. In this study, we used transcriptome sequencing to compare newly diagnosed MM patients who had short progression- free survival (PFS) to standard first-line treatment (PFS < 24 months) to patients who had prolonged PFS (PFS > 24 months). We identified 157 differentially upregulated lncRNAs with short PFS and focused our efforts on characterizing the most upregulated lncRNA, LINC01432. We investigated LINC01432 overexpression and CRISPR/Cas9 knockdown in MM cell lines to show that LINC01432 overexpression significantly increases cell viability and reduces apoptosis, while knockdown significantly reduces viability and increases apoptosis, supporting the clinical relevance of this lncRNA. Next, we used individual-nucleotide resolution cross-linking immunoprecipitation with RT-qPCR to show that LINC01432 directly interacts with the RNA binding protein, CELF2. Lastly, we showed that LINC01432-targeted locked nucleic acid antisense oligonucleotides reduce viability and increases apoptosis. In summary, this fundamental study identified lncRNAs associated with short PFS to standard NDMM treatment and further characterized LINC01432, which inhibits apoptosis. Key points: lncRNA expression was found to be dysregulated in patients with short PFS to standard multiple myeloma therapy. LINC01432-bound CELF2 inhibits apoptosis.

cancer biology↗

bioRxiv2023

High-density volumetric super-resolution microscopy

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce temporal resolution by requiring lower labelling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMFLM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax. We then experimentally demonstrate the high accuracy (>99.2 {+/-} 0.1%, 0.1 locs m-2) and sensitivity (>86.6 {+/-} 0.9%, 0.1 locs m-2) of SMLFM at point detection through whole-cell (scan-free) imaging and tracking of single membrane proteins in live primary B cells. We also exemplify high density volumetric imaging (0.15 locs m-2) in dense cytosolic tubulin datasets.

biophysics↗

bioRxiv2023

POLCAM: Instant molecular orientation microscopy for the life sciences

Current methods for single-molecule orientation localization microscopy (SMOLM) require optical setups and algorithms that can be prohibitively slow and complex, limiting the widespread adoption for biological applications. We present POLCAM, a simplified SMOLM method based on polarized detection using a polarization camera, that can be easily implemented on any wide-field fluorescence microscope. To make polarization cameras compatible with single-molecule detection, we developed theory to minimize field of view errors, used simulations to optimize experimental design, and developed a fast algorithm based on Stokes parameter estimation which can operate over 1000 fold faster than the state of the art, enabling near instant determination of molecular anisotropy. To aid in the adoption of POLCAM, we developed open-source image analysis software, and a website detailing hardware installation and software use. To illustrate the potential of POLCAM in the life sciences, we applied our method to study alpha-synuclein fibrils, the actin cytoskeleton of mammalian cells, fibroblast-like cells and the plasma membrane of live human T cells.

biophysics↗

bioRxiv2021

Comparative analysis of single nucleotide polymorphisms and microsatellite markers for parentage verification and discovery within the equine Thoroughbred breed

Short tandem repeat (STR), also known as microsatellite markers are currently used for genetic parentage verification within equine. Transitioning from STR to single nucleotide polymorphism (SNP) markers to perform equine parentage verification is now a potentially feasible prospect and a key area requiring evaluation is parentage testing accuracies when using SNP based methods, in comparison to STRs. To investigate, we utilised a targeted equine genotyping by sequencing (GBS) panel of 562 SNPs to SNP genotype 309 Thoroughbred horses - inclusive of 55 previously parentage verified offspring. Availability of STR profiles for all 309 horses, enabled comparison of parentage accuracies between SNP and STR panels. An average sample call rate of 97.2% was initially observed, and subsequent removal of underperforming SNPs realised a pruned final panel of 516 SNPs. Simulated trio and partial parentage scenarios were tested across 12-STR, 16-STR, 147-SNP and 516-SNP panels. False-positives (i.e. expected to fail parentage, but pass) ranged from 0% for 147-SNP and 516-SNP panels to 0.003% when using 12-STRs within trio parentage scenarios, and 0% for 516-SNPs to 1.6% for 12-STRs within partial parentage scenarios. Our study leverages targeted GBS methods to generate low-density equine SNP profiles and demonstrates the value of SNP based equine parentage analysis in comparison to STRs - particularly when performing partial parentage discovery.

genetics↗

bioRxiv2020

Food waste as a resource: grinding, dilution, and storage as a pretreatment strategy to produce fermentation intermediates

In several states of the U.S., one measure to mitigate greenhouse-gas emissions has been to ban food wastes from landfills. As a result, U.S.-based companies are now providing decentralized food-waste management systems for supermarkets and restaurants, which include storage as a slurry. It is unclear, however, which storage conditions (factors) would affect the spontaneous microbial activity, resulting in a different fermentation product spectra, and how this would affect further post-treatment. Here, we performed two experiments to mimic: 1) storage and 2) subsequent anaerobic digestion. For the food-waste storage system, we designed a mixed-level fractional factorial analysis with 12 experimental combinations, including separating food waste into: carbohydrate-rich, lipid-rich, and protein-rich food waste. We found that all factors that we tested correlated with the fermentation product spectra, but that especially the factors: i) storage temperature; ii) food-waste composition; and iii) storage time affected the fermentation outcome. We observed that relatively low pH levels of 3-4, which were achieved due to rapid lactic acid accumulation by microbial activity during storage, coincided with greater lactate production at a maximum chemical oxygen demand (COD) selectivity of 90%. This provides an opportunity to optimize lactate production, which is ideal for subsequent methane or chemical production. TOC/Abstract graphic O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

bioengineering↗