Biology subjects

Ouellet, J.

Publications and source records attributed to Ouellet, J..

8 recordsLinked to original sources

bioRxiv2026

PARP1/2-linked ADP-ribosylation impairs trophoblast mitochondrial respiration in a TNF-α model of inflammation-mediated preeclampsia.

Inflammation-mediated preeclampsia (I-PE) is associated with increased placental NAD+-dependent ADP-ribosylation and NAD+ depletion. We investigated whether PARP1/2- or tankyrase-sensitive ADP-ribosylation contributes to inflammation-associated trophoblast dysfunction and whether pharmacological inhibition improves trophoblast function under inflammatory stress. We profiled mRNA expression of genes encoding enzymes that catalyze poly-ADP-ribosylation (PARP1, PARP2, TNKS1, TNKS2) and selected enzymes that catalyze mono-ADP-ribosylation (PARP7, PARP10, PARP12, and PARP14), in human I-PE placentas (control n=23; I-PE n=10) and an LPS-induced rat model of I-PE (control n=8, LPS n=8). HTR-8/SVneo cells were treated with TNF- alone or with olaparib, (PARP1/2 inhibitor) or XAV939 (TNKS1/2 inhibitor). Global mono- and poly-ADP-ribosylation (MAR/PAR), including a PARP1-associated ~120-kDa ADP-ribosylation signal and pathway enrichment of proteins identified from this region, were assessed alongside mitochondrial respiration, OXPHOS protein abundance, 4-hydroxynonenal, migration, and viability. In human I-PE placentas, mRNA abundance of poly-ADP-ribosylating enzymes did not differ significantly from controls, whereas mono-ADP-ribosylating enzymes were increased in LPS-exposed rat placentas. In HTR-8/SVneo cells, TNF- induced a prominent PARP1 associated ~120-kDa MAR/PAR signal that was suppressed by olaparib, but not XAV939. Proteins identified from this ~120-kDa region were enriched for known PARP1 targets and immune/stress-response pathways. Olaparib, but not XAV939, improved basal and maximal respiration, ATP production, and spare respiratory capacity. TNF- did not significantly alter oxidative phosphorylation (OXPHOS) subunits, 4-HNE abundance, viability, or migration at 24 h. These data support PARP1/2-sensitive ADP-ribosylation, most consistent with PARP1-linked activity, as a contributor to inflammation-mediated trophoblast bioenergetic dysfunction. The PARP1/2-NAD+ axis may therefore represent a mechanism-directed target for future studies of I-PE.

cell biology↗

bioRxiv2025

Senolytic Therapy as a Preventive Strategy for Low Back Pain

Cell senescence drives inflammation and tissue breakdown and is a key hallmark of aging. Low back pain is strongly linked to age-related degeneration of spine tissues, and with an accumulation of senescent. Here we show that preventive administration of the senolytic agents o-vanillin and RG-7112 prevent the development of pain-related behaviour in young sparc-/- mice. Treated mice exhibit a reduction of senescence markers in the intervertebral discs, vertebral endplates, vertebral bone, and spinal cord, alongside a dampening of pro-inflammatory senescence-associated secretory factors in these tissues. This early senolytic intervention also preserves intervertebral disc volume and vertebral bone microarchitecture, indicating protection against structural degeneration of the spine. These findings demonstrate that targeting cellular senescence at an early stage can mitigate degenerative changes and pain, supporting senolytic therapy as a promising preventive strategy for musculoskeletal decline.

cell biology↗

bioRxiv2025

Beyond miRNA cargo profiles: anti-inflammatory roles of extracellular vesicle-enriched miRNAs derived from human intervertebral disc cells unveiled by functional testing

Intervertebral disc (IVD) degeneration is a leading cause of chronic low back pain and a major contributor to global disability. Understanding the molecular mechanisms underlying this condition is essential for developing targeted therapies. Among these mechanisms, microRNAs (miRNAs) have emerged as critical post-transcriptional regulators of gene expression in IVD cells, influencing key processes such as extracellular matrix (ECM) homeostasis, inflammatory signalling, and cellular senescence. Extracellular vesicles (EVs), which transport miRNAs between cells, represent a promising avenue for therapeutic intervention. However, the composition of their miRNA cargo across different stages of disc degeneration remains inadequately characterized. We isolated EVs from primary human IVD cells derived from non-degenerate, mildly-degenerate, and severely degenerate tissues, and performed small RNA sequencing to profile their miRNA content. Bioinformatic analyses revealed enrichment in pathways related to ECM-receptor interaction, focal adhesion, inflammation, and cell cycle regulation. Notably, let-7b-5p and miR-100-5p were among the most abundant miRNAs and were significantly lower in EVs from degenerate discs. Functional assays demonstrated that transfection of IVD cells with let-7b-5p or miR-100-5p mimics individually suppressed IL-1{beta} expression at both mRNA and protein levels, confirming their anti-inflammatory roles. Strikingly, co-delivery of both miRNAs enhanced suppression of pro-inflammatory mediators, reduced senescence-associated p16 expression, and upregulated TIE2 mRNA, indicating synergistic effects in promoting a regenerative cell phenotype. These findings highlight the regulatory roles of EV-enriched let-7b-5p and miR-100-5p in modulating inflammation and senescence in IVD cells, and underscore the potential of miRNA-loaded EVs as cell-free regenerative therapies for disc degeneration.

cell biology↗

bioRxiv2024

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

biochemistry↗

bioRxiv2022

Shifted Reverse PAGE: a novel approach based on structure switching for the discovery of riboswitches and aptamers

Riboswitches are regulatory sequences composed of an aptamer domain capable of binding a ligand and an expression platform that allows the control of the downstream gene expression based on a conformational change. Current bioinformatic methods for their discovery have various limitations. To circumvent this, we developed an experimental technique to discover new riboswitches called SR-PAGE (Shifted Reverse Polyacrylamide Gel Electrophoresis). A ligand-based regulatory molecule is recognized by exploiting the conformational change of the sequence following binding with the ligand within a native polyacrylamide gel. Known riboswitches were tested with their corresponding ligands to validate our method. SR-PAGE was imbricated within an SELEX to enrich switching RNAs from a TPP riboswitch-based degenerate library to change its binding preference from TPP to thiamine. The SR-PAGE technique allows performing a large screening for riboswitches, search in several organisms and test more than one ligand simultaneously.

molecular biology↗

bioRxiv2021

Folding and Persistence Time of Intramolecular G-Quadruplexes Transiently Embedded in a DNA duplex

G-quadruplex (G4) DNA structures have emerged as important regulatory elements during DNA replication, transcription or repair. While many in-vitro studies have focused on the kinetics of G4 formation within DNA single-strands, G4 are found in-vivo in double-stranded DNA regions, where their formation is challenged by pairing between the two complementary strands. Since the energy of hybridization of Watson-Crick structures dominates the energy of G4 folding, this competition should play a critical role on the persistence of G4 in vivo. To address this issue, we designed a single molecule assay allowing measuring G4 folding and persistence while the structure is periodically challenged by the complementary strand. We quantified both the folding rate and the persistence time of biologically relevant G4 structures and showed that the dynamics of G4 formation depends strongly on the genomic location. G4 are found much more stable in promoter regions and replication origins than in telomeric regions. In addition, we characterized how G4 dynamics was affected by G4 ligands and showed that both folding rate and persistence increased. Our assay opens new perspectives for the measurement of G4 dynamics, which is critical to understand their role in genetic regulation.

biophysics↗

bioRxiv2020

Single-cell RNA sequencing reveals time- and sex-specific responses of spinal cord microglia to peripheral nerve injury and links ApoE to neuropathic pain

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unknown whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. We show, using single-cell RNA-sequencing, that nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in males as compared to females. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following injury. Apolipoprotein E (Apoe) is the top upregulated gene in microglia at chronic time points after nerve injury in mice and polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a previously unrecognized role for ApoE in neuropathic pain.

neuroscience↗

bioRxiv2020

Detecting genetic variation and base modifications together in the same single molecules of DNA and RNA at base pair resolution using a magnetic tweezer platform.

Accurate decoding of nucleic acid variation is important to understand the complexity and regulation of genome function. Here we introduce a single-molecule platform based on magnetic tweezer (MT) technology that can identify and map the positions of sequence variation and multiple base modifications together in the same single molecules of DNA or RNA at single base resolution. Using synthetic templates, we demonstrate that our method can distinguish the most common epigenetic marks on DNA and RNA with high sensitivity, specificity and precision. We also developed a highly specific CRISPR-Cas enrichment strategy to target genomic regions in native DNA without amplification. We then used this method to enrich native DNA from E. coli and characterized the differential levels of adenine and cytosine base modifications together in molecules of up to 5 kb in length. Finally, we enriched the 5 UTR of FMR1 from cells derived from a Fragile X carrier and precisely measured the repeat expansion length and methylation status of each molecule. These results demonstrate that our platform can detect a variety of genetic, epigenetic and base modification changes concomitantly within the same single molecules.

genomics↗