Biology subjects

Bernard, S.

Publications and source records attributed to Bernard, S..

11 recordsLinked to original sources

bioRxiv2026

MR-Guided Focused Ultrasound Neuromodulation of Nucleus Accumbens to Attenuate Oral Fentanyl Motivation in A Self-Administration Rat Model

Introduction: Transcranial low-intensity focused ultrasound (LIFU) is being explored as a non-invasive approach for neuromodulation to treat opioid use disorder (OUD). Methods: We evaluated MR-guided focused ultrasound, with LIFU, targeting nucleus accumbens (MRgLIFU-NAc) bilaterally, using MR-acoustic radiation force-imaging (MR-ARFI) to ensure accurate targeting and adjust for FUS intensity delivered at the target. Male and female rats were trained to self-administer fentanyl/sucrose (70ug/ml fentanyl citrate in 5% sucrose) or 5% sucrose alone. Three cohorts of rats (total n=76) were evaluated on progressive ratio (PR) responding for oral fentanyl/sucrose at either 4 or 24 hours post-MRgLIFU-NAc. Animals in the first two cohorts received repeated rounds of LIFU or sham (anesthesia-duration matched). In cohort 3, we used a counterbalanced within-subjects design in which rats received both LIFU and sham one week apart. Results: Reducing sucrose concentrations decreased responding for sucrose alone more than for fentanyl/sucrose, demonstrating a role for fentanyl in the reinforcing properties of fentanyl/sucrose. Across three sonication schemes, MRgLIFU-NAc did not attenuate PR performance for oral fentanyl/sucrose at 4 or 24-hours post-LIFU relative to controls. Adjusting LIFU intensity with MR-ARFI did not correlate with changes in PR from baseline. We confirmed that NAc is the correct target by bilateral inactivation of NAc with muscimol/baclofen mixture (50 + 50 ng/side), which reduced PR responding for fentanyl/sucrose. Conclusion: The three sets of LIFU parameters, with repeated MRgLIFU-NAc sessions, did not produce detectable changes in oral fentanyl/sucrose motivation. Further studies are required to determine effective LIFU parameters needed to reduce NAc-dependent fentanyl motivation.

neuroscience↗

bioRxiv2026

A survey of multi-targeting and off-targeting sgRNAs across five genome-wide CRISPR-Cas9 knockout sgRNA libraries with GuideRefine

Pooled genome-wide CRISPR-Cas9 knockout (CRISPR-KO) screening is a powerful approach for discovering new biology and identifying genetic vulnerabilities in cancers. This approach uses the Cas9 nuclease in combination with sgRNA libraries, typically consisting of 4-8 sgRNAs to induce mutations in each target gene. A critical assumption is that the effect of each sgRNA is solely due to Cas9 editing of the target gene. However, libraries can contain sgRNAs that direct Cas9 to multiple locations, thus potentially introducing bias into gene hit lists and leading to flawed biological hypotheses. Here we have developed GuideRefine, a pipeline to detect multi-targeting and off-targeting sgRNAs. GuideRefine outputs a virtual refined sub-library containing only on-target sgRNAs. Using GuideRefine with T2T-CHM13 as the reference genome, we surveyed the Brunello, TKOv3, Yusa, Avana, and Jacquere libraries, finding that ~7.5% to ~16% of sgRNAs are potentially problematic. We confirmed that multi-targeting sgRNAs disproportionately impair cell fitness and that sgRNAs aligning to more than one location with a single mismatch can also reduce fitness, although to a lesser extent. After flagging problematic sgRNAs and creating virtual ''on-target only'' sub-libraries, ~10% to ~16% of genes lose critical representation (< 3 sgRNAs per gene). Intriguingly, a set of 467 genes, characterised by short CDS length and lower PAM site density, have fewer than three sgRNAs in all sub-libraries, suggesting they cannot be well-targeted using current libraries. We anticipate that GuideRefine, together with caution in assessing the effects of problematic sgRNAs, will help prioritise biologically relevant hits.

bioinformatics↗

bioRxiv2026

IMAGENE: Single-cell association of live cell imaging and gene expression profiles of non-adherent cells through photoactivatable adhesives

Live cell imaging is uniquely placed to study cell behavior as it preserves spatial context and enables non-destructive observations over time. Integrating live cell imaging and molecular phenotypes with single-cell resolution is key to uncovering the relationship between the behavioral and morphological signatures of cells, and their molecular states. Non-adherent cells - as are most immune cells - however, present unique challenges in linking live cell imaging and fixed cell assays with single-cell resolution due to the difficulty of identifying individual cells across experimental modalities. To overcome this issue, we developed IMAGENE, an experimental and computational pipeline that leverages previously reported photoactivatable biocompatible adhesive material (PA-BAM) coatings for on-the-fly cell immobilization. We demonstrate the IMAGENE experimental and computational pipeline by generating a dataset of label-free time-lapse videos of primary human naive CD8+ T cells following 24 hours of polyclonal stimulation. Individual cells, including highly motile cells, can be matched to expression profiles of genes of interest obtained through KrakenFISH, a modified version of the previously reported autoFISH setup for automated, single-molecule fluorescence in situ hybridization (smFISH) experiments that supports sample parallelization. We use this data to train explainable machine learning models that predict expression levels of individual genes, with variable performance, from hand-crafted dynamic and spatial features obtained from live cell imaging.

bioengineering↗

bioRxiv2025

CDC7 and CDK8 kinases cooperate to support DNA replication origin firing in human cells

The coordinated activation of DNA replication origins is important for efficient DNA synthesis and genome stability. S-phase cyclin dependent kinases (CDKs) together with CDC7 kinase, are essential to origin activation by converting the pre-replicative complex into a fully active helicase. To identify genes that tune DNA replication, we have performed a chemo-genetic genome-wide CRISPR-KO screen with cells challenged with the CDC7 inhibitor XL413. By developing a methodology based on genetic coessentiality to functionally cluster the hits, we uncover the transcriptional CDK8/CCNC kinase in a cluster with replication initiation factors. We find that CDK8 depletion further reduces the rate of DNA synthesis imposed by CDC7 inhibitors. DNA fibre experiments provide compelling evidence that CDK8 and CDC7 cooperate in origin activation. Suppression of DNA synthesis by CDK8 inhibition requires the binding of CDK8/CCNC to the MDM2 Binding Protein (MTBP) and we show that CDC7 and CDK8 individually contribute to the phosphorylation of MCM4 subunit of the replicative helicase. Thus, this work identifies CDK8 as the third protein kinase directly involved in origin activation in human cells.

biochemistry↗

bioRxiv2025

Multiscale modeling of the spatial structure of stem cells in neuroblastoma patient-derived tumoroids reveals a critical role for a short-range diffusive process

Neuroblastomas are heterogeneous pediatric tumors of the sympathetic nervous system for which treatments are still limited. Fundamental and applied approaches have been enabled thanks to the generation of patient-derived tumoroids (PDTs), ex vivo 3D structures used as avatars of the original tumor. We generated neuroblastoma PDTs and quantified the spatial distribution of CD133+ cancer stem cells using immunohistochemistry. We observed that those cells tend to aggregate in the PDT. In order to better understand the set of rules needed for generating such structures, we implemented a multiscale agent-based neuroblastoma tumoroid model. Model rules specify single cells fate based on its intracellular content, which dynamically evolves according to a stochastic gene regulatory network. The state of this network can be modulated by cell-to-cell signalling through neighbor cells fate decisions and, possibly, spatial location. We first observed that in the absence of any spatial rules for inter-cellular interactions, no spatial structure emerged. The addition of simple rules (signalling by cell-to-cell contact or differential cell adhesion) only marginally improved the quantitative agreement to the experimental dataset. In sharp contrast, the addition of short-range pro-stem cell diffusive signalling among stem cells produced very realistic 3D PDT-like structures. This works highlights the power of our multiscale approach to discard too simplistic rules and to propose a minimal set of hypotheses required to reproduce qualitatively and quantitatively experimentally observed spatial structures. In the case of neuroblastomas-derived PDTs, short-range spatial diffusion of stem-to-stem cell signalling proved to play a key role in successfully reconstructing the spatial structure.

cancer biology↗

bioRxiv2024

Reversible and reusable compartmentalized microfluidic chip for coculture of dorsal root ganglion neurons

Compartmentalized microfluidic chips play an important role in understanding the cellular mechanisms involved in neurodegenerative disorders. Dorsal root ganglia are a well-established model for modelling the peripheral nervous system (PNS), but their development on a chip remains limited. Furthermore, it would be beneficial for the devices to be openable in order to access the biological material inside for analyses. Easy to prototype and biocompatible, styrenic block copolymers (SBC) are an alternative to polydimethylsiloxane (PDMS) that offer both reversible and permanent bonding properties. This paper presents a fast and straightforward method to produce compartmentalized SBC chips. The study validates the culture of murine dorsal root ganglia explants, comparing it to the standard methods, to obtain a model of the PNS. Moreover, the reversible bonding properties of the SBC permit the reuse of the chip with a quick and easy cleaning protocol. It provides direct access to the cells, opening the way for imaging and molecular biology analysis. The comparison of the resources required to produce PDMS and SBC chips highlights the importance of moving to reusable devices. These detachable, easy-to-manufacture and sustainable all-thermoplastic platforms provide an alternative way of prototyping compartmentalized devices for in vitro PNS modeling.

biophysics↗

bioRxiv2024

Effect of Alcohol and Cocaine Abuse on Neuronal and Non-Neuronal Cell Turnover in the Adult Human Hippocampus

Clinical studies on humans with a history of chronic abuse of alcohol or cocaine show cognitive impairments associated with hippocampal atrophy. Adult hippocampal neurogenesis is a process important for memory formation and has been shown to be impaired by alcohol and cocaine in rodent models. It has thus been suggested that a reduction in adult neurogenesis may contribute to cognitive dysfunctions seen in patients with abuse. In addition, reduced adult neurogenesis has been suggested to play a role in the pathology of addiction vulnerability. We have previously demonstrated persistent adult hippocampal neurogenesis throughout life by measuring 14C concentrations in genomic DNA, incorporated during cell division, in a mixed cohort of subjects. In this study, we use the same strategy to assess the extent of cell turnover of neuronal and non-neuronal cells in the hippocampus of humans with known history of alcohol and cocaine abuse and compare these with healthy controls. We find that there is significant neuronal and non-neuronal turnover in healthy controls, as well as in individuals with long term alcohol use orcocaine use. Using mathematical modelling, we compare the extent of cell turnover of neurons and non-neuronal cells and did not find any significant difference between healthy controls and the two addiction groups. While we cannot exclude scenarios of altered adult neurogenesis over shorter periods of time, our data does not support the theory of low neurogenesis as a mechanism of addiction vulnerability.

neuroscience↗

bioRxiv2024

In silico modelling of CD8 T cell immune response links genetic regulation to population dynamics

The CD8 T cell immune response operates at multiple temporal and spatial scales, including all the early complex biochemical and biomechanical processes, up to long term cell population behavior. In order to model this response, we devised a multiscale agent-based approach using Simuscale software. Within each agent (cell) of our model, we introduced a gene regulatory network (GRN) based upon a piecewise deterministic Markov process (PDMP) formalism. Cell fate - differentiation, proliferation, death - was coupled to the state of the GRN through rule-based mechanisms. Cells interact in a 3D computational domain and signal to each other via cell-cell contacts, influencing the GRN behavior. Results show the ability of the model to correctly capture both population behaviour and molecular time-dependent evolution. We examined the impact of several parameters on molecular and population dynamics, and demonstrated the add-on value of using a multiscale approach by showing that a higher degradation rate for the protein controlling cell death induces a later peak in the response.

systems biology↗

bioRxiv2020

Evidence for motility in 3.4 Gyr-old organic-walled microfossils ?

The oldest traces for planktonic lifestyle have been reported in ca. 3.4 billion years old silicified sediments from the Strelley Pool Formation in Western Australia. Observation of flange appendages suggests that Archean life motility was passive and driven by drifting of microorganisms in their surrounding environment. Until now, the oldest traces for active motility are ca. 2.1 billion years old. Whether or not active motility already existed during the Archean eon remains an open question. Here we report the discovery of new 3.4 billion years old tailed microfossils. These microfossils exhibit a lash-like appendage that likely provided them with movement capabilities. This suggests that these microfossils are the oldest remains of active motile life forms. With the ability to move in liquids and on organic and/or mineral surfaces, these microorganisms were capable of escaping from harsh environments and/or colonizing new ecological niches as early as 3.4 billion years ago. The existence of these deep-rooted Archean motile life forms offers a new picture of the Archean biodiversity, with unanticipated evolutionary innovative morphological complexities.

paleontology↗

bioRxiv2020

Phenotypic Noise and the Cost of Complexity

Experimental studies demonstrate the existence of phenotypic diversity despite constant genotype and environment. Theoretical models based on a single phenotypic character predict that during an adaptation event, phenotypic noise should be positively selected far from the fitness optimum because it increases the fitness of the genotype, and then be selected against when the population reaches the optimum. It is suggested that because of this fitness gain, phenotypic noise should promote adaptive evolution. However, it is unclear how the selective advantage of phenotypic noise is linked to the rate of evolution, and whether any advantage would hold for more realistic, multi-dimensional phenotypes. Indeed, complex organisms suffer a cost of complexity, where beneficial mutations become rarer as the number of phenotypic characters increases. By using a quantitative genetics approach, we first show that for a one-dimensional phenotype, phenotypic noise promotes adaptive evolution on plateaus of positive fitness, independently from the direct selective advantage on fitness. Second, we show that for multi-dimensional phenotypes, phenotypic noise evolves to a low-dimensional configuration, with elevated noise in the direction of the fitness optimum. Such a dimensionality reduction of the phenotypic noise promotes adaptive evolution and numerical simulations show that it reduces the cost of complexity.

evolutionary biology↗

bioRxiv2019

Cell Generation Dynamics Underlying Naïve T Cell Homeostasis in Adult Humans

Thymic involution and proliferation of naive T cells both contribute to shaping the naive T cell repertoire as humans age, but a clear understanding of the roles of each throughout a human lifespan has been difficult to determine. By measuring nuclear bomb test-derived 14C in genomic DNA we determined the turnover rates of CD4+ and CD8+ naive T cell populations and defined their dynamics in healthy individuals ranging from 20-65 years of age. We demonstrate that naive T cell generation decreases with age, and that this could be explained by a combination of declining cell loss, peripheral division and thymic production during adulthood. We investigated putative mechanisms underlying age-related changes in homeostatic regulation of naive T cell turnover using mass cytometry to profile candidate signaling pathways involved in T cell activation and proliferation in CD4+ naive T cells relative to CD31 expression, a marker of thymic proximity. We show that basal NF-{kappa}B phosphorylation inversely correlated with CD31 expression and thus is decreased in peripherally expanded naive T cell clones. Functionally we found that NF-{kappa}B signaling was essential for naive T cell proliferation to the homeostatic growth factor IL-7, and reduced NF-{kappa}B phosphorylation in CD4+CD31- naive T cells is linked to reduced homeostatic proliferation potential. Our results reveal an age-related decline in naive T cell turnover as a putative regulator of naive T cell diversity and identify a molecular pathway that restricts proliferation of peripherally expanded naive T cell clones that accumulate with age.

immunology↗