Biology subjects

Gauthier, L.

Publications and source records attributed to Gauthier, L..

11 recordsLinked to original sources

bioRxiv2026

CM/Pf contribution to mesocircuit dynamics underlying awareness in non-human primates

BackgroundIn acute disorders of consciousness following severe brain injury, the heterogeneity and multiplicity of brain lesions make it difficult to infer a direct causal link between lesion patterns and impaired consciousness. As a result, neuroimaging analyses performed in intensive care settings can inform theories of consciousness mainly through correlational, rather than causal, evidence. The mesocircuit hypothesis proposes that interactions between the thalamus, basal ganglia, and cortex support the neural mechanisms underlying awareness. Among its key components, the intralaminar thalamic nuclei are thought to play a central role, yet their respective contributions to mesocircuit dynamics remain poorly understood. MethodsIn two macaque monkeys, we combined causal perturbations of three thalamic nuclei (the centromedian-parafascicular complex (CM/Pf), central lateral nucleus (CL), and mediodorsal nucleus (MD)) with a cue-guided behavioural task requiring the processing of multisensory information. Electrical microstimulation was first used to compare the behavioural consequences of perturbing each target. Reversible pharmacological inactivation using muscimol was subsequently performed in the CM/Pf and CL. Finally, the large-scale metabolic effects of CM/Pf inactivation (muscimol) and disinhibition (bicuculline) were assessed using [18F]FDG PET imaging. ResultsPerturbation of the intralaminar nuclei disrupted behavioural performance, with the strongest and most reproducible effects observed following CM/Pf manipulation. CM/Pf inactivation reduced correct responses and increased omissions and wrong responses, particularly under conditions requiring greater behavioural engagement. Importantly, these deficits occurred without evidence of major wakefulness impairment, as animals remained responsive throughout testing and showed no substantial increase in eye-closure probability. In contrast, CL perturbation produced weaker behavioural effects, suggesting distinct functional contributions of the two intralaminar nuclei. [18F]FDG PET imaging revealed that CM/Pf disinhibition increased glucose metabolism across thalamic, striatal, pallidal, and frontal cortical territories, whereas CM/Pf inactivation induced a broadly reciprocal pattern of metabolic pattern. These findings identify the CM/Pf as a major modulator of both thalamo-striatal and thalamo-cortical components of the mesocircuit. ConclusionsOur results show that a unilateral intralaminar nucleus perturbation is not sufficient to affect wakefulness, suggesting that this function is mediated by a bilateral network at the thalamus level within the mesocircuit. The CM/Pf emerged as the intralaminar nucleus exerting the strongest influence on goal directed behaviours requiring the mesocircuit dynamics. Our findings also support the existence of partially distinct intralaminar pathways contributing differentially to global state regulation of awareness-related functions, and identify the CM/Pf as a key thalamo-striatal hub within the mesocircuit.

neuroscience↗

bioRxiv2026

Overcoming Protein A-driven Nonspecific Antibody Staining of S. aureus in Immunofluorescence Microscopy

The ability to detect host cell factors during Staphylococcus aureus infection in vitro by immunofluorescence microscopy is severely hampered by staphylococcal protein A (SpA), a cell wall-anchored protein that binds the fragment crystallizable (Fc) region of immunoglobulins. This interaction generates strong nonspecific fluorescent signals on the bacterial surface, complicating data interpretation and limiting the accuracy of quantitative image analysis. Several measures have been put forward to overcome this obstacle, most importantly the pre-incubation with an anti-SpA antibody (SpA) and the use of human serum (HS) as blocking agent and antibody diluent. To highlight this feature to general fluorescence microscopy users, we here systematically evaluated these two strategies. Using S. aureus coated on coverslips and S. aureus-infected A549 cells, we highlight the efficiencies of both approaches to markedly reduce nonspecific fluorescence, with HS treatment yielding the most profound suppression. Notably, HS, containing high levels of human immunoglobulins, offered a robust, cost-effective and broadly applicable solution for minimizing SpA-driven artifacts, thereby improving immunofluorescence microscopy in S. aureus infection models in vitro.

microbiology↗

bioRxiv2025

A strategy for genome-wide seamless tagging of human protein-coding genes

Comprehensive and systematic proteome-wide experiments require financial and technical resources unavailable to most researchers. Here we describe a scalable CRISPR/Cas9-non-homologous end joining (NHEJ) based method for Pooled Recombinant Integration of Seamless Markers (PRISM) into protein coding genes. We created two gRNA libraries for 5- and 3-tagging of 18,804 human protein-coding genes. Selection for in-frame integration of the donor cassette can be guaranteed by fusing it to an antibiotic resistance enzyme (ARE) and P2A self-cleaving peptide, resulting in tagged proteins and free ARE to select for clones with inserted donor cassettes. We achieved a library integration rate of 19.75% and tagging of [~]80% for genes expressed in Hek293T cells and notably, 89.7% of essential genes, with donor DNA. Our strategy is scalable, specific, and selective, paving the way for genome-scale construction of human cell lines tagged with different types of reporter genes for protein functional characterization. Significance statementHere we report the first practical method to achieve near complete 5- or 3-end integration of reporter protein-coding sequences to express seamless fusions of human protein and reporter proteins that we call Pooled Recombinant Integration of Seamless Markers (PRISM). PRISM is independent of homology templates, yet it works with high efficiency and precision. Using gRNA-directed CRISPR/Cas9 genome editing and mini-plasmid donor cassettes we were able to tag about 80% of the protein-coding sequences of human genes with a green florescent protein at an integration efficiency of about 20% and most notably, of 90% of essential genes in the human cell line HEK 293T. This is important because the characterization of gene functions in a particular cell type usually begins with essential genes.

systems biology↗

bioRxiv2024

Genome-wide CRISPR/Cas9 screen reveals factors that influence the susceptibility of tumor cells to NK cell-mediated killing

BackgroundNatural killer (NK) cells exhibit potent cytotoxic activity against various cancer cell types. Over the past five decades, numerous methodologies have been employed to elucidate the intricate molecular mechanisms underlying NK cell-mediated tumor control. While significant progress has been made in elucidating the interactions between NK cells and tumor cells, the regulatory factors governing NK cell-mediated tumor cell destruction are not yet fully understood. This includes the diverse array of tumor ligands recognized by NK cells and the mechanisms that NK cells employ to eliminate tumor cells. MethodsIn this study, we employed a genome-wide CRISPR/Cas9 screening approach in conjunction with functional cytotoxicity assays to delineate the proteins modulating the susceptibility of colon adenocarcinoma HCT-116 cells to NK cell-mediated cytotoxicity. ResultsAnalysis of guide RNA (gRNA) distribution in HCT-116 cells that survived co-incubation with NK cells identified ICAM-1 as a pivotal player in the NKp44-mediated immune synapse, with NKp44 serving as an activating receptor crucial for the elimination of HCT-116 tumor cells by NK cells. Furthermore, disruption of genes involved in the apoptosis or IFN-{gamma} signaling pathways conferred resistance to NK cell attack. We further dissected that NK cell-derived IFN-{gamma} promotes mitochondrial apoptosis in vitro and exerts control over B16-F10 lung metastases in vivo. ConclusionMonitoring ICAM-1 levels on the surface of tumor cells or modulating its expression should be considered in the context of NK cell-based therapy. Additionally, considering the diffusion properties of IFN-{gamma}, our findings highlight the potential of leveraging NK cell-derived IFN-{gamma} to enhance direct tumor cell killing and facilitate bystander effects via cytokine diffusion, warranting further investigation. WHAT IS ALREADY KNOWN ON THIS TOPICNK cells play a crucial role in identifying and eliminating various cancer cell types. However, the mechanisms that regulate NK cell-mediated destruction of tumor cells are not yet fully understood. This involves the array of tumor ligands that NK cells recognize and the processes they utilize to carry out tumor cell elimination. WHAT THIS STUDY ADDSOur research emphasizes the critical role of ICAM-1 in NKp44-mediated destruction of HCT-116 tumor cells. Additionally, we found that interfering with genes related to apoptosis or IFN-{gamma} signaling pathways increased resistance to NK cell attack. We showed that IFN-{gamma} produced by NK cells induces mitochondrial apoptosis in vitro and helps regulate B16-F10 lung metastases in vivo. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICYGiven the ability of IFN-{gamma} to diffuse, our findings suggest that NK cell-derived IFN-{gamma} can be harnessed to directly kill tumor cells and trigger bystander effects through cytokine spread. This approach holds promise for further exploration. Additionally, assessing or manipulating ICAM-1 levels on tumor cell surfaces could enhance the effectiveness of NK cell-based therapies.

immunology↗

bioRxiv2023

Genetic landscape of an in vivo protein interactome

Protein-protein interaction (PPI) networks accurately map environmental perturbations to their molecular consequences in cells, but effects of genome-wide genetic variation on PPIs remain unknown. We hypothesized that PPI networks integrate genetic and environmental effects, potentially defining biochemical mechanisms underlying complex polygenic traits. Here, we measured 61 PPIs in inbred strains of Saccharomyces cerevisiae with [~]12,000 single-nucleotide polymorphisms (SNPs) across the genome. Unlike mRNA expression and protein abundance that are primarily affected by SNPs local (in "cis") to a gene, PPIs are predominantly affected by SNPs far (in "trans") to the genomic loci of the interacting proteins. However, consistent with the PPI networks small-world characteristic, these transacting SNPs are in neighboring genes in the network. We likewise discovered SNPs in non-coding RNAs and post-transcriptional regulators (3 UTRs) with, counterintuitively, larger PPI-modulating effects than SNPs within protein-coding regions. Finally, we inferred known and novel mechanisms of action for yeast and human drugs. HIGHLIGHTSO_LIProtein-interaction quantitative trait locus ("piQTL") mapping reveals sensitivity of in vivo PPIs to polymorphisms across the yeast genome C_LIO_LITrans-piQTLs significantly outnumber and are stronger than cis-piQTLs C_LIO_LISNPs in non-coding RNAs and 3 UTRs have comparable effects to PPI as SNPs in coding regions C_LIO_LIpiQTL mapping reveals known and novel mechanism of yeast and human drugs C_LI

genomics↗

bioRxiv2022

Intra- and inter-species interactions drive early phases of invasion in mice gut microbiota

The stability and dynamics of ecological communities are dictated by interaction networks typically quantified at the level of species.1-10 But how such networks are influenced by intra-species variation (ISV) is poorly understood.11-14 Here, we use ~500,000 chromosomal barcodes to track high-resolution intra-species clonal lineages of Escherichia coli invading mice gut with the increasing complexity of gut microbiome: germ-free, antibiotic-perturbed, and innate microbiota. By co-clustering the dynamics of intra-species clonal lineages and those of gut bacteria from 16S rRNA profiling, we show the emergence of complex time-dependent interactions between E. coli clones and resident gut bacteria. With a new approach, dynamic covariance mapping (DCM), we differentiate three phases of invasion in susceptible communities: 1) initial loss of community stability as E. coli enters; 2) recolonization of some gut bacteria; and 3) recovery of stability with E. coli coexisting with resident bacteria in a quasi-steady state. Comparison of the dynamics, stability and fitness from experimental replicates and different cohorts suggest that phase 1 is driven by mutations in E. coli before colonization, while phase 3 is by de novo mutations. Our results highlight the transient nature of interaction networks in microbiomes driven by the persistent coupling of ecological and evolutionary dynamics. One-Sentence SummaryHigh-resolution lineage tracking and dynamic covariance mapping (DCM) define three distinct phases during early gut microbiome invasion.

evolutionary biology↗

bioRxiv2022

A genome-wide mutational constraint map quantified from variation in 76,156 human genomes

The depletion of disruptive variation caused by purifying natural selection (constraint) has been widely used to investigate protein-coding genes underlying human disorders, but attempts to assess constraint for non-protein-coding regions have proven more difficult. Here we aggregate, process, and release a dataset of 76,156 human genomes from the Genome Aggregation Database (gnomAD), the largest public open-access human genome reference dataset, and use this dataset to build a mutational constraint map for the whole genome. We present a refined mutational model that incorporates local sequence context and regional genomic features to detect depletions of variation across the genome. As expected, proteincoding sequences overall are under stronger constraint than non-coding regions. Within the non-coding genome, constrained regions are enriched for known regulatory elements and variants implicated in complex human diseases and traits, facilitating the triangulation of biological annotation, disease association, and natural selection to non-coding DNA analysis. More constrained regulatory elements tend to regulate more constrained protein-coding genes, while non-coding constraint captures additional functional information underrecognized by gene constraint metrics. We demonstrate that this genome-wide constraint map provides an effective approach for characterizing the non-coding genome and improving the identification and interpretation of functional human genetic variation.

genetics↗

bioRxiv2021

Mitochondrial DNA variation across 56,434 individuals in gnomAD

Databases of allele frequency are extremely helpful for evaluating clinical variants of unknown significance; however, until now, genetic databases such as the Genome Aggregation Database (gnomAD) have ignored the mitochondrial genome (mtDNA). Here we present a pipeline to call mtDNA variants that addresses three technical challenges: (i) detecting homoplasmic and heteroplasmic variants, present respectively in all or a fraction of mtDNA molecules, (ii) circular mtDNA genome, and (iii) misalignment of nuclear sequences of mitochondrial origin (NUMTs). We observed that mtDNA copy number per cell varied across gnomAD cohorts and influenced the fraction of NUMT-derived false-positive variant calls, which can account for the majority of putative heteroplasmies. To avoid false positives, we excluded samples prone to NUMT misalignment (few mtDNA copies per cell), cell line artifacts (many mtDNA copies per cell), or with contamination and we reported variants with heteroplasmy greater than 10%. We applied this pipeline to 56,434 whole genome sequences in the gnomAD v3.1 database that includes individuals of European (58%), African (25%), Latino (10%), and Asian (5%) ancestry. Our gnomAD v3.1 release contains population frequencies for 10,850 unique mtDNA variants at more than half of all mtDNA bases. Importantly, we report frequencies within each nuclear ancestral population and mitochondrial haplogroup. Homoplasmic variants account for most variant calls (98%) and unique variants (85%). We observed that 1/250 individuals carry a pathogenic mtDNA variant with heteroplasmy above 10%. These mitochondrial population allele frequencies are publicly available at gnomad.broadinstitute.org and will aid in diagnostic interpretation and research studies.

genomics↗

bioRxiv2019

Chromosomal barcoding of E. coli populations reveals lineage diversity dynamics at high resolution

Evolutionary dynamics in large asexual populations is strongly influenced by multiple competing beneficial lineages, most of which segregate at very low frequencies. However, technical barriers to tracking a large number of these rare lineages have so far prevented a detailed elucidation of evolutionary dynamics in large bacterial populations. Here, we overcome this hurdle by developing a chromosomal barcoding technique that allows simultaneous tracking of [~]450,000 distinct lineages in E. coli. We used this technique to gather insights into the evolutionary dynamics of large (>107 cells) E. coli populations propagated for [~]420 generations in the presence of sub-inhibitory concentrations of common antibiotics. By deep sequencing the barcodes, we reconstructed trajectories of individual lineages at high frequency resolution (< 10-5). Using quantitative tools from ecology, we found that populations lost lineage diversity at distinct rates corresponding to their antibiotic regimen. Additionally, by quantifying the reproducibility of these dynamics across replicate populations, we found that some lineages had similar fates over independent experiments. Combined with an analysis of individual lineage trajectories, these results suggest how standing genetic variation and new mutations may contribute to adaptation to sub-inhibitory antibiotic levels. Altogether, our results demonstrate the power of high-resolution barcoding in studying the dynamics of bacterial evolution.

evolutionary biology↗

bioRxiv2018

Stepwise evolution and convergent recombination underlie the global dissemination of carbapenemase-producing Escherichia coli

Carbapenem-resistant Enterobacteriaceae are considered by WHO as \"critical\" priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates, as well as the genetic bases for the emergence and global dissemination of specific lineages, remain largely unknown. Here, by combining a thorough genomic and evolutionary analysis of Ec ST410 isolates with a broad analysis of 12,584 E. coli and Shigella genomes, we showed that the fixation of carbapenemase genes depends largely on a combination of mutations in ftsI encoding the penicillin binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele inducing reduced susceptibility to diverse {beta}-lactams spread across the species by recombination. The selection of CP-Ec lineages able to disseminate is more complex than the mere acquisition of carbapenemase genes.

microbiology↗

bioRxiv2017

SodaPop: A computational suite for simulating the dynamics of asexual populations

MotivationSimulating protein evolution with realistic constraints from population genetics is essential in addressing problems in molecular evolution, from understanding the forces shaping the evolutionary landscape to the clinical challenges of antibiotic resistance, viral evolution and cancer.\n\nResultsTo address this need, we present SodaPop, a new forward-time simulator of large asexual populations aimed at studying their structure, dynamics and the distribution of fitness effects with flexible assumptions on the fitness landscape. SodaPop integrates biochemical and biophysical properties in a cell-based, object-oriented framework and provides an efficient, open-source toolkit for performing large-scale simulations of protein evolution.\n\nAvailability and implementationSource code and binaries are freely available at https://github.com/louisgt/SodaPop under the GNU GPLv3 license. The software is implemented in C++ and supported on Linux, Mac OS/X and Windows.\n\nContactadrian.serohijos@umontreal.ca\n\nSupplementary informationSupplementary information is available on the Github project page.

bioinformatics↗