Biology subjects

Rousseau, A.

Publications and source records attributed to Rousseau, A..

12 recordsLinked to original sources

bioRxiv2026

Morphologic intratumoral heterogeneity from routine whole-slide histopathology is prognostic for survival in primary central nervous system lymphoma: development in the LOC Network and international external validation

Background: Clinical scores incompletely capture outcomes in primary central nervous system lymphoma (PCNSL). We quantified morphologic heterogeneity in pretreatment hematoxylin and eosin (H\&E) whole slides. Patients and methods: Three independent cohorts of immunocompetent, HIV- and EBV-negative patients treated recently were analyzed: LOC 2023 (122 slides), phase III BLOCAGE-01 (245 slides; NCT02313389), and external Barcelona (BCN; 41 slides). UNI embeddings, prototype learning, spatial metrics, and elastic-net Cox regression defined ITH-C. Results: Models achieved bootstrap-corrected concordance of 0.797--0.834. Age-, sex-, and KPS-adjusted ITH-C HRs were 1.29 (95\% CI 1.01--1.64), 1.27 (1.07--1.51), and 2.13 (1.35--3.37), respectively. Adding ITH-C increased MSKCC C-index from 0.671 to 0.717, 0.560 to 0.593, and 0.588 to 0.706. Spatial transcriptomics linked ITH-C to immune programs. Conclusions: Routine H\&E encodes prognostic spatial heterogeneity in PCNSL. ITH-C complements clinical scores, supporting prospective risk stratification.

bioinformatics↗

bioRxiv2026

Functional diversity of Vγ9Vδ2 T cells overcomes glioblastoma state plasticity and antigen heterogeneity

Glioblastoma (GBM) is characterized by a high degree of cellular plasticity and intra-tumoral heterogeneity, which frequently leads to the failure of standard therapies, including immunotherapies. While chimeric antigen receptor (CAR) T cells offer a potent means of MHC-independent tumor recognition, their efficacy is hampered by the coexistence of distinct molecular states, gathered as proneural (PN) and mesenchymal (MES) phenotypes. Here, we demonstrate that gangliosides GD2 and O-acetylated GD2 (OAcGD2) are preferentially expressed by PN cells whereas MES cells display reduced expression due to upregulated ganglioside catabolism. Conversely, MES cells are known to exhibit high expression of stress-induced ligands recognized by V{gamma}9V{delta}2 T cells. We show that while engineering V{delta}2T cells with GD2- or OAcGD2-specific CAR enables the elimination of PN cells, it also facilitates the immune escape of MES cells in heterogeneous 3D-tumoroid models. Mechanistically, we reveal a hierarchy of receptor engagement, where CAR signaling predominates leading to the structural and functional exclusion of endogenous TCR from the immunological synapse. To address this receptor competition, we propose a strategy that leverages the functional effector diversity by combining untransduced and CAR-engineered V{delta}2T cells. This dual approach provides a dynamic safety net by ensuring the simultaneous elimination of PN and MES cells and preventing the selective outgrowth of resistant cells. Our findings establish a conceptual framework for designing off-the-shelf immunotherapies tailored to the metabolic and phenotypic plasticity of resistant solid tumors.

cancer biology↗

bioRxiv2026

Exploration of targeted electrophilic kinase probes identifies a covalent ULK1 degrader

Kinases have proven to be one of the most fertile target classes for new drug approvals. However, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we systematically assessed the impact of minimal electrophilic modifications to ATP-site binding scaffolds, leading us to identify molecules that can control the activity and abundance of the master autophagy regulator, Unc-51-like autophagy activating kinase 1 (ULK1).

biochemistry↗

bioRxiv2025

Characterization of HIV-1 particles co-purified with three extracellular vesicle subtypes from the Raji CD4 DCIR cell line, a hybrid model of CD4 T cells and dendritic cells

BackgroundHIV-1 proteins and RNA are packaged into extracellular vesicles (EVs) through interaction with the multivesicular endosomes of the EV biogenesis machinery. This interaction also allows functional or abortive viruses to exit the cells as exosomes, microvesicles or apoptotic vesicles. HIV-1 viral particles and EVs share significant similarities in size, composition, and molecular cargo, making their separation challenging. Current HIV-1 purification methods neglect the effects of EVs on virus infectivity, which could influence experiment outcomes. Here, we co-characterized HIV-1 particles co-purified with exosomes, microvesicles and apoptotic vesicles to determine their impact on HIV-1 infection. MethodsThe HIV-infected Raji CD4 DCIR cells supernatants were harvested 2 and 8 days after infection. The 2-day supernatant was treated with proteinase K to discard viral protein and HIV-1 RNA associated with proteins outside the EVs. The supernatants were fractionated into three pellets by differential centrifugation. The 3K pellet contained the largest EVs, such as apoptotic vesicles. The 17K and 100K pellets were respectively associated with microvesicles and exosomes. EVs and viral particles were co-characterized for their host and viral contents and the pellets obtained after 8 days post-infection were tested for infectivity. ResultsProteinase K treatment notably lowered HIV-1 RNA concentration in the EV pellet and did not affect viral p24 capsid protein concentration. The p24 protein was mostly found in the 17K pellet and HIV-1 RNA was the most abundant in the 100K pellet for both 2- and 8-day productions. Nevertheless, the 3K pellet had the highest infectivity when cells were infected with an equal quantity of virus (measured by p24) from each pellet. ConclusionProductively infected cells released functional viruses in the three EV subtypes, each exerting a distinct effect on virus infectivity. For future experiments, the presence of EVs in viral preparations should be taken into account, as they influence the progression of HIV-1 infection. HighlightsO_LIHIV-1 RNA and p24 capsid protein are packaged within apoptotic vesicles, microvesicles and exosomes released by Raji CD4 DCIR cells. C_LIO_LIInfectious particles co-precipitated with the 3K pellet from Raji CD4 DCIR cells are more infectious than other viruses associated with other EV pellets. C_LI

cell biology↗

bioRxiv2023

Distinct TORC1 signalling branches regulate Adc17 proteasome assembly chaperone expression

When stressed, cells need to adapt their proteome to maintain protein homeostasis. This requires increased proteasome assembly. Increased proteasome assembly is dependent on increased proteasome assembly chaperone expression. In S. cerevisiae, inhibition of the growth-promoting kinase complex TORC1 causes increased proteasome assembly chaperone expression. This is dependent upon activation of the MAPKinase Mpk1 and relocalisation of assembly chaperone mRNA to patches of dense actin. We show here that TORC1 inhibition alters cell wall properties to induce these changes by activating the Cell Wall Integrity pathway through the Wsc1, Wsc3, and Wsc4 sensor proteins. We demonstrate that in isolation these signals are insufficient to drive protein expression. We identify that the TORC1-activated S6Kinase Sch9 must be inhibited as well. This work expands our knowledge on the signalling pathways which regulate proteasome assembly chaperone expression. SummaryTORC1-regulated proteasome assembly chaperone expression is necessary for proteasome assembly and proteostasis. We identify Cell Wall Integrity and Sch9 signalling as mechanisms controlling this.

cell biology↗

bioRxiv2023

Multimodal mucosal and systemic immune characterization of a novel non-human primate trachoma model highlights the critical role of local immunity during acute phase disease

BackgroundTrachoma -the leading cause of blindness worldwide as a result of infection-is caused by repeated Chlamydia trachomatis (Ct) conjunctival infections. Disease develops in two phases: i) active (acute trachoma, characterized by follicular conjunctivitis), then long-term ii) scarring (chronic trachoma, characterized by conjunctival fibrosis, corneal opacification and eyelid malposition). Scarring trachoma is driven by the number and the severity of reinfections. The immune system is a pivotal aspect of disease, involved in disease aggravation, but also key for exploitation in development of a trachoma vaccine. Therefore, we characterized clinical and local immune response kinetics in a non-human primate model of acute conjunctival Ct infection and disease. Methodology/Principal FindingsThe conjunctiva of non-human primate (NHP, Cynomolgus monkeys -Macaca fascicularis-) were inoculated with Ct (B/Tunis-864 strain, B serovar). Clinical ocular monitoring was performed using a standardized photographic grading system, and local immune responses were assessed using multi-parameter flow cytometry of conjunctival cells, tear fluid cytokines, immunoglobulins, and Ct quantification. Clinical findings were similar to those observed during acute trachoma in humans, with the development of typical follicular conjunctivitis from the 4th week post-exposure to the 11th week. Immunologic analysis revealed an early phase influx of T cells in the conjunctiva and elevated interleukins 4, 8, and 5, before a later phase monocytic influx accompanied by a decrease in other immune cells, and tear fluid cytokines returning to initial levels. Conclusion/SignificanceOur NHP model accurately reproduces acute trachoma clinical signs, allowing for the precise assessment of the local immune responses in infected eyes. A progressing immune response occurred for weeks after exposure to Ct, which subsided into persistence of innate immune responses. Understanding these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies to prevent disease. Author SummaryChlamydia trachomatis is the leading infectious cause of blindness worldwide. The pathogenesis of trachoma is more complicated than other types of bacterial conjunctivitis: clinical signs of trachoma are rooted in repeated Chlamydia trachomatis infections of the inner eyelid surfaces, which roughens the skin. This lead to eyelid deformation and lashes rubbing on the cornea, which across multiple years of abrasion, ends with corneal opacification. The immune system is a pivotal aspect of disease, involved in disease aggravation, but also key for exploitation in development of a trachoma vaccine. Here we describe a non-human primate model of trachoma that accurately reproduces acute human eye disease, allowing for the precise assessment of the local immune responses in infected eyes. A progressing immune response occurred 4 weeks after exposure to Ct, which subsided into persistence of innate immune responses. Understanding these local responses is the first step towards using the model to assess new vaccine and therapeutic strategies to prevent disease.

microbiology↗

bioRxiv2023

Characterizing fibrinolysis under flow conditions: a simplified mesoscale 3D model

One of the routine clinical treatments to eliminate ischemic stroke thrombi is injecting a biochemical product into the patients bloodstream, which breaks down the thrombis fibrin fibers: intravenous or intravascular thrombolysis. However, this procedure is not without risk for the patient; the worst circumstances can cause a brain hemorrhage or embolism that can be fatal. Improvement in patient management drastically reduced these risks, and patients who benefited from thrombolysis soon after the onset of the stroke have a significantly better 3-month prognosis, but treatment success is highly variable. The causes of this variability remain unclear, and it is likely that some fundamental aspects still require thorough investigations. For that reason, we conducted in vitro flow-driven fibrinolysis experiments to study pure fibrin thrombi breakdown in controlled conditions and observed that the lysis front evolved non-linearly in time. To understand these results, we developed an analytical 1D lysis model in which the thrombus is considered a porous medium. The lytic cascade is reduced to a second-order reaction involving fibrin and a surrogate pro-fibrinolytic agent. The model was able to reproduce the observed lysis evolution under the assumptions of constant fluid velocity and lysis occurring only at the front. For adding complexity, such as clot heterogeneity or complex flow conditions, we propose a 3-dimensional mesoscopic numerical model of blood flow and fibrinolysis, which validates the analytical models results. Such a numerical model could help us better understand the spatial evolution of the thrombi breakdown, extract the most relevant physiological parameters to lysis efficiency, and possibly explain the failure of the clinical treatment. These findings suggest that even though real-world fibrinolysis is a complex biological process, a simplified model can recover the main features of lysis evolution.

biophysics↗

bioRxiv2023

Genetic screening identifies integrated stress response kinase HRI (EIF2AK1) as a negative regulator of PINK1 and mitophagy signalling

Loss-of-function mutations of the PINK1 kinase cause familial early-onset Parkinsons disease (PD). PINK1 is activated upon mitochondrial damage to phosphorylate Ubiquitin and Parkin to trigger removal of damaged mitochondria by autophagy (mitophagy). PINK1 also indirectly phosphorylates a subset of Rab GTPases including Rab8A. We have performed an siRNA screen targeting all human Ser/Thr kinases in HeLa cells and discovered that knockdown of the eukaryotic translation initiation factor 2-alpha kinase 1 (EIF2AK1), also known as heme-regulated inhibitor (HRI) kinase, a branch of the integrated stress response (ISR), selectively enhances mitochondrial depolarization-induced stabilization of PINK1 and increased phosphorylation of ubiquitin and Rab8A. We confirm our findings in multiple human cell lines, including SK-OV-3, U2OS and ARPE-19 cells. Knockdown of the upstream mitochondrial-cytosol relay component, DELE1, enhanced PINK1 stabilisation and activation similar to EIF2AK1 knockdown. Strikingly, we demonstrate that the small molecule ISR inhibitor, ISRIB, also enhances PINK1 activation and signaling under conditions of mitochondrial damage. Using the mito-QC mitophagy reporter in human cells, we observe that EIF2AK1 knockdown or ISRIB treatment significantly enhances PINK1-dependent mitophagy but does not alter deferiprone-induced mitophagy. Our findings indicate that the DELE1-EIF2AK1 ISR signaling relay is a negative regulator of PINK1-dependent mitophagy and suggest that inhibitors of DELE1-EIF2AK1 and/or ISRIB analogues could have therapeutic benefits in PD and related disorders.

biochemistry↗

bioRxiv2022

The ribosome-associated chaperone Zuo1 controls translation upon TORC1 inhibition

Eukaryotic cells achieve proteostasis by ensuring their protein requirements are met through tight control of TORC1 activity. Upon TORC1 inhibition, degradative activity is increased, and protein synthesis is reduced through inhibition of translation initiation, to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system is required to maintain proteostasis and cell viability under TORC1 inhibition, in yeast. In the absence of the Hsp40 cochaperone Zuo1, translation does not decrease in response to loss of TORC1 activity. The functional interaction between Zuo1 and its Hsp70 partner, Ssb, is required for proper translational control and proteostasis maintenance upon TORC1 inhibition. Further, we have found that the rapid degradation of eIF4G following TORC1 inhibition is prevented in zuo1{Delta} cells, contributing to decreased survival in these conditions. Our findings suggest a new role for RAC/Ssb in regulating translation in response to changes in TORC1 signalling.

molecular biology↗

bioRxiv2022

Actin remodelling controls proteasome homeostasis upon stress

When cells are stressed, bulk translation is often downregulated to reduce energy demands whilst stress-response proteins are simultaneously upregulated. 19S Regulatory Particle Assembly-Chaperones (RPACs) are selectively translated upon TORC1 inhibition to promote proteasome assembly and activity, maintaining cell viability. However, the molecular mechanism for such selective translational upregulation is unclear. Using yeast, we discover that remodelling of the actin cytoskeleton is important for RPAC translation following TORC1 inhibition. mRNA of the RPAC ADC17 travels along actin cables and is enriched at cortical actin patches under stress, dependent upon the early endocytic protein Ede1. ede1{Delta} cells failed to induce RPACs and proteasome assembly upon TORC1 inhibition. Conversely, artificially tethering ADC17 mRNA to cortical actin patches enhanced its translation upon stress. These findings suggest that actin dense structures such as cortical actin patches may serve as a translation platform for a subset of stress-induced mRNAs including regulators of proteasome homeostasis.

cell biology↗

bioRxiv2021

Investigating the two regimes of fibrin clot lysis: an experimental and computational approach

It has been observed in vitro that complete clot lysis is generally preceded by a period of latency during which the degradation seems to be inefficient. However, this latency was merely notified but not yet quantitatively discussed. In our experiments we observed that the lysis ubiquitously occurred in two distinct regimes, a slow and a fast lysis regime. We quantified extensively the duration of these regimes for a wide spectrum of experimental conditions and found that on average the slow regime lasts longer than the fast one, meaning that during most of the process the lysis is ineffective. We proposed a computational model in which the two regimes result from a spatially constrained kinetic of clot lysis: first the biochemical reactions take place at the outer core of the fibrin fibers composing the clot, then in the bulk resulting in the observed fast lysis regime. This simple hypothesis appeared to be sufficient to reproduce with a great accuracy the lysis profiles obtained experimentally. Our results shed light on new insights regarding the dynamical aspects of the lysis of fibrin rich clots in a context where the timing is so critical for patient treatment and outcome. SignificanceWhile the interplay between the main components of the fibrinolytic system is well understood, some dynamical aspects of the fibrinolysis remain unclear. Notably, we observe that in vitro fibrin rich clots undergo a slow and inefficient phase of degradation when subject to endogenous fibrinolysis. In fact, it turns out that a large part of the lysis process operates in this slow regime. To explain this observation, we proposed a computational model in which the properties of the binding of the proteins change during the lysis. First plasminogen and tissue plasminogen activator bind at the surface of the fibers, resulting in a slow lysis, then in the bulk of the fibers thus speeding up the degradation of the clot..

biophysics↗

bioRxiv2017

Promyelocytic Leukemia (PML) Nuclear Bodies (NBs) Induce Latent/Quiescent HSV-1 Genomes Chromatinization Through a PML-NB/Histone H3.3/H3.3 Chaperone Axis

Herpes simplex virus 1 (HSV-1) latency establishment is tightly controlled by promyelocytic leukemia (PML) nuclear bodies (NBs) (or ND10), although their exact implication is still elusive. A hallmark of HSV-1 latency is the interaction between latent viral genomes and PML-NBs, leading to the formation of viral DNA-containing PML-NBs (vDCP-NBs). Using a replication-defective HSV-1-infected human primary fibroblast model reproducing the formation of vDCP-NBs, combined with an immuno-FISH approach developed to detect latent/quiescent HSV-1, we show that vDCP-NBs contain both histone H3.3 and its chaperone complexes, i.e., DAXX/ATRX and HIRA complex (HIRA, UBN1, CABIN1, and ASF1a). HIRA also co-localizes with vDCP-NBs present in trigeminal ganglia (TG) neurons from HSV-1-infected wild type mice. ChIP-qPCR performed on fibroblasts stably expressing tagged H3.3 (e-H3.3) or H3.1 (e-H3.1) show that latent/quiescent viral genomes are chromatinized almost exclusively with e-H3.3, consistent with an interaction of the H3.3 chaperones with multiple viral loci. Depletion by shRNA of single proteins from the H3.3 chaperone complexes only mildly affects H3.3 deposition on the latent viral genome, suggesting a compensation mechanism. In contrast, depletion (by shRNA) or absence of PML (in mouse embryonic fibroblast (MEF) pml-/- cells) significantly impacts the chromatinization of the latent/quiescent viral genomes with H3.3 without any overall replacement with H3.1. Consequently, the study demonstrates a specific epigenetic regulation of latent/quiescent HSV-1 through an H3.3-dependent HSV-1 chromatinization involving the two H3.3 chaperones DAXX/ATRX and HIRA complexes. Additionally, the study reveals that PML-NBs are major actors in latent/quiescent HSV-1 H3.3 chromatinization through a PML-NB/histone H3.3/H3.3 chaperone axis.\n\nAuthor summaryAn understanding of the molecular mechanisms contributing to the persistence of a virus in its host is essential to be able to control viral reactivation and its associated diseases. Herpes simplex virus 1 (HSV-1) is a human pathogen that remains latent in the PNS and CNS of the infected host. However, the latency is unstable, and frequent reactivations of the virus are responsible for PNS and CNS pathologies. It is thus crucial to understand the physiological, immunological and molecular levels of interplay between latent HSV-1 and the host. Promyelocytic leukemia (PML) nuclear bodies (NBs) play a major role in controlling viral infections by preventing the onset of lytic infection. In previous studies, we showed a major role of PML-NBs in favoring the establishment of a latent state for HSV-1. A hallmark of HSV-1 latency establishment is the formation of PML-NBs containing the viral genome, which we called \"viral DNA-containing PML-NBs\" (vDCP-NBs). The genome entrapped in the vDCP-NBs is transcriptionally silenced. This naturally occurring latent/quiescent state could, however, be transcriptionally reactivated. Therefore, understanding the role of PML-NBs in controlling the establishment of HSV-1 latency and its reactivation is essential to design new therapeutic approaches based on the prevention of viral reactivation.

microbiology↗