Biology subjects

Richard, A.

Publications and source records attributed to Richard, A..

12 recordsLinked to original sources

bioRxiv2026

Cornealia: automated collective order analysis reveals density-dependent reorganization of donor corneal endothelium

The corneal endothelium is a non-regenerative monolayer whose health determines transplant success, yet donor tissue is assessed only by single-cell metrics that ignore how the monolayer is organized collectively. A physics-based spring constant K, derived from the radial distribution function of cell positions, captures this collective order but has remained limited to semi-manual studies without physical interpretation. Here we present Cornealia, an automated pipeline for collective-order and intercellular-force analysis. In cultured endothelial cells, collective order declines with passaging and is higher under growth-factor conditions that preserve N-cadherin junctions, differences invisible to cell density and hexagonality. Across 2,177 donor cornea images from two cohorts, collective order, distinct from classical metrics, responds most to mechanical perturbation: corneas with endothelial cell density above 2,400 cells/mm^2 mostly gain order, whereas less dense ones lose it, on one curve shared by both cohorts. Reconstructed intercellular forces track these changes. This switch lies within the minimum donor densities used by eye banks, suggesting a mechanical basis for these clinical thresholds and establishing collective order as an automatable measure of endothelial quality.

bioinformatics↗

bioRxiv2026

Transient immune landscape remodelling shapes CD8 T-cell priming during infection

Immune responses to intracellular pathogens require the generation of a CD8 T-cell response comprised of clones with a broad range of T Cell Receptor (TCR) affinities for their cognate peptide. While high-affinity clones clear pathogens during initial infection, low-affinity T-cells are critical to protect against subsequent encounters with mutated pathogens. Low-affinity T-cells are recruited to an immune response despite known mechanisms that limit their expansion and priming. As such, the mechanism safeguarding the recruitment of low-affinity T-cell clones during infection remains unclear. Here, we show that at the start of an infection, the spatial immune architecture is transiently reorganised to initiate broad T-cell priming. In particular, Tregs become segregated from the initial priming site, resulting in a spatio-temporal window during which pathogen-specific T-cell priming is protected from Treg inhibition. Priming induces the expression of CXCR3 in a TCR affinity-dependent manner, which conditions the retention of activated T-cells in the priming region. Inability to express CXCR3 leads to T-cell relocation towards Treg-rich areas, where their expansion is limited by the lack of the costimulatory molecule CD70. Our study redefines the role of Treg during infection and provides a mechanism to explain how the priming and breadth of the CD8 T-cell response is protected during infection.

immunology↗

bioRxiv2025

Sequence and ionic requirements of pUG fold quadruplexes

Poly(UG) or "pUG" repeat RNA can fold into a left-handed parallel quadruplex, the pUG fold. The pUG fold directs the amplification of RNAi in C. elegans, and pUG sequences are abundant in eukaryotic transcriptomes. Here, we report the sequence and ionic requirements for pUG folding. The pUG fold requires 12 guanosines but has an otherwise flexible sequence requirement. The uridines can be substituted with other nucleotides, with some sequence variants folding better than pUG RNA. The GA repeat sequence (GA)12 also forms a pUG-like fold, albeit with lower thermodynamic stability than (GU)12. The pUG fold can tolerate multiple deoxyribose substitutions but does not fold when the backbone is entirely deoxyribose. It has a high affinity and specificity for potassium ions (K1/2 = 6 mM) and does not fold in sodium or ammonium ions. Addition of 2 mM Mg2+ does not further stabilize the pUG fold, and the polyamines spermine and spermidine slightly decrease its stability. Finally, the pUG fold is sensitive to surrounding sequence context and complementary flanking sequences can stabilize pUG folds, while other sequences can interfere with folding. These data expand our understanding of RNA folding and provide a basis for understanding and predicting pUG folds.

biochemistry↗

bioRxiv2025

Variation in virion phosphatidylserine content drives differential GAS6 binding among closely related flaviviruses

Many enveloped viruses engage phosphatidylserine (PS) receptors to enter cells, a phenomenon known as "apoptotic mimicry". We previously reported that Zika virus (ZIKV), but not closely-related West Nile virus (WNV) or dengue virus (DENV), utilized AXL to infect cells because only ZIKV could bind the AXL ligand GAS6, a PS-binding protein. In this study, we investigated the mechanisms underlying the differential ability of these viruses to bind GAS6. Although immature virions expose larger patches of the viral membrane than do mature ones, our data show that virion maturity levels did not contribute to GAS6 binding. Surprisingly, while ZIKV contains PS comparable to cellular membranes, PS on WNV and DENV is markedly reduced. These findings explain why only ZIKV can bind GAS6 and provide insights into a novel mechanism by which closely-related flaviviruses differentially utilize cellular entry factors. IMPORTANCEAmong flaviviruses, Zika virus uniquely causes microcephaly and congenital defects. While no flavivirus-specific entry receptors have been identified, they commonly take advantage of phosphatidylserine (PS) receptors to enter cells. Our previous studies revealed that Zika virus uniquely utilizes AXL, found in immune-privileged sites such as the brain and placenta, via binding to its ligand, GAS6. Our current study shows that despite being produced from the same cells, Zika virus has substantially higher PS content than closely related dengue and West Nile viruses, which likely explains Zika viruss unique ability to bind GAS6. These findings provide insight into how closely related flaviviruses can vary substantially in their use of cellular entry factors, potentially contributing to the distinct diseases they cause.

microbiology↗

bioRxiv2025

RIPOR2 promotes multinucleation of melanoma cells downstream of the RAS/ERK oncogenic pathway

One-third of skin melanomas arise from melanocytic nevi, benign skin lesions composed of clustered melanocytes. Benign nevi are associated with overactivation of the mitogen-activated protein kinase RAS/ERK pathway, resulting from driver mutations, most commonly in the BRAF or NRAS gene. However, this overactivation in melanocytes is insufficient to induce melanoma formation, as only a minority of benign nevi give rise to melanoma. Overactivation of the RAS/ERK pathway promotes genetic and epigenetic alterations by inducing aneuploidy, but the processes by which nevi evolve into melanoma via RAS/ERK pathway-dependent aneuploidy are only partially understood. Using single-nucleus RNA sequencing after overactivation of the RAS/ERK pathway in the chicken embryo, we discovered that RIPOR2 is a positive transcriptional target of this pathway, including in melanocyte precursors. Similar transcriptional control of RIPOR2 by RAS/ERK is conserved in human melanoma cells. RIPOR2 emerged as an attractive target because it encodes an atypical RHOA inhibitory protein involved in the development of physiologically multinucleated cell types. Multinucleation in cancer has been shown to promote aneuploidy, which correlates with tumor aggressiveness. We found that RIPOR2 is ectopically expressed in human nevi and skin melanomas and functionally promotes multinucleation in both an animal model and human tumor-derived cells, including melanoma cell lines. Our results suggest that RIPOR2 expression, downstream of RAS/ERK overactivation in skin melanocytes, promotes the emergence of multinucleated cells, a previously overlooked step in melanoma formation.

cancer biology↗

bioRxiv2024

Emergence of Pre-movement Beta Activity with Stable Sensorimotor Predictions to Facilitate Motor Adjustments

Adaptive behavior enables flexible responses to environmental changes. This process is particularly crucial when transitioning between environments with different features, relying on the progressive formation of expectations based on prior experience. In humans, beta oscillations are central to adaptive behavior. Yet, the brain mechanisms underlying the detection of environmental changes, and the iterative update needed to progressively improve behavioral performance remain elusive. Here, we reveal that beta activity emerges in a cerebello-cortical network two seconds before action initiation, as the features of a new environment become known and behavioral outcomes become more predictable. Within this period, the cerebellum and parietal cortex drive prefrontal activity to form expectations. Using a single-trial approach, we establish that beta bursts before action initiation predict performance in the upcoming trial based on previous outcomes. These findings uncover a novel anticipatory mechanism that reflects predictive processes critical for stabilizing performance and adapting to environmental changes.

neuroscience↗

bioRxiv2024

The ability of human TIM1 to bind phosphatidylethanolamine enhances viral uptake and efferocytosis compared to rhesus and mouse orthologs

T-cell Immunoglobulin and Mucin (TIM)-family proteins facilitate the clearance of apoptotic cells, are involved in immune regulation, and promote infection of enveloped viruses. These processes are frequently studied in experimental animals such as mice or rhesus macaques, but functional differences among the TIM orthologs from these species have not been described. Previously, we reported that while all three human TIM proteins bind phosphatidylserine (PS), only human TIM1 (hTIM1) binds phosphatidylethanolamine (PE), and that this PE-binding ability contributes to both phagocytic clearance of apoptotic cells and virus infection. Here we show that rhesus macaque TIM1 (rhTIM1) and mouse TIM1 (mTIM1) bind PS but not PE and that their inability to bind PE makes them less efficient than hTIM1. We also show that alteration of only two residues of mTIM1 or rhTIM1 enables them to bind both PE and PS, and that these PE-binding variants are more efficient at phagocytosis and mediating viral entry. Further, we demonstrate that the mucin domain also contributes to the binding of the virions and apoptotic cells, although it does not directly bind phospholipid. Interestingly, contribution of the hTIM1 mucin domain is more pronounced in the presence of a PE-binding head domain. These results demonstrate that rhTIM1 and mTIM1 are inherently less functional than hTIM1, owing to their inability to bind PE and their less functional mucin domains. They also imply that mouse and macaque models underestimate the activity of hTIM1. SIGNIFICANCEWe previously reported that human T-cell Immunoglobulin and Mucin protein 1 (TIM1) binds phosphatidylethanolamine (PE) as well as phosphatidylserine (PS) and that PE is exposed on the apoptotic cells and viral envelopes. Moreover, TIM1 recognition of PE contributes to phagocytic clearance of apoptotic cells and virus uptake. Here we report that unlike human TIM1, murine and rhesus TIM1 orthologs bind only PS, and as a result, their ability to clear apoptotic cells or promote virus infection is less efficient. These findings are significant because they imply that the activity of TIM1 in humans is greater than what the studies conducted in common animal models would indicate.

molecular biology↗

bioRxiv2024

Multi-Niche Human Bone Marrow On-A-Chip for Studying the Interactions of Adoptive CAR-T Cell Therapies with Multiple Myeloma

Multiple myeloma (MM), a cancer of bone marrow plasma cells, is the second-most common hematological malignancy. However, despite immunotherapies like chimeric antigen receptor (CAR)-T cells, relapse is nearly universal. The bone marrow (BM) microenvironment influences how MM cells survive, proliferate, and resist treatment. Yet, it is unclear which BM niches give rise to MM pathophysiology. Here, we present a 3D microvascularized culture system, which models the endosteal and perivascular bone marrow niches, allowing us to study MM-stroma interactions in the BM niche and model responses to therapeutic CAR-T cells. We demonstrated the prolonged survival of cell line-based and patient-derived multiple myeloma cells within our in vitro system and successfully flowed in donor-matched CAR-T cells. We then measured T cell survival, differentiation, and cytotoxicity against MM cells using a variety of analysis techniques. Our MM-on-a-chip system could elucidate the role of the BM microenvironment in MM survival and therapeutic evasion and inform the rational design of next-generation therapeutics. TEASERA multiple myeloma model can study why the disease is still challenging to treat despite options that work well in other cancers.

cancer biology↗

bioRxiv2019

Erythroid differentiation displays a peak of energy consumption concomitant with glycolytic metabolism rearrangements

It has been suggested that a switch from glycolysis, with lactate production, toward mitochondrial oxidative phosphorylation (OXPHOS) could be a driving force during stem cell differentiation.\n\nBased upon initial results, from our previous work, showing a drop in LDHA mRNA level during the differentiation of chicken erythroid progenitors, we studied metabolism behavior to question whether such switch might also be operating in those cells.\n\nWe first analyzed the level of 9 enzymes, including LDHA, involved either in glycolysis or OXPHOS, in self-renewing and differentiating cells. Our results suggest that erythroid differentiation might be accompanied by an enhancement of the respiratory chains and glycolysis activities at 12h, followed by a strong decline of the glycolytic pathway and a stabilization of OXPHOS.\n\nTo confirm that OXPHOS might be increased and glycolysis decreased during erythroid differentiation, we measured lactate concentration and mitochondrial membrane potential (MMP) of self-renewing and differentiating cells. Our findings show that at 12h-24h of differentiation, a surge of energy is needed, which could be fueled jointly by glycolysis and OXPHOS. Then the energy demand comes back to normal and might be supplied by OXPHOS instead of lactate production through glycolysis.\n\nThese results support the hypothesis that erythroid differentiation is associated with a metabolic switch from glycolysis to OXPHOS.\n\nWe also assessed LDHA role in erythroid progenitors self-renewal and the metabolic status changes. Inhibition experiments showed that LDHA activity could be involved in the maintenance of erythroid progenitors self-renewal, and its decline could influence their metabolic status.\n\nFinally, we investigated whether these metabolic rearrangements were necessary for erythroid differentiation. The addition of an inhibitor of the respiratory chains affected progenitors ability to differentiate, suggesting that the metabolic switch from glycolysis toward OXPHOS might act as a driving force for erythroid differentiation.\n\nAuthor summarySingle-cell based gene expression data from one of our previous publication pointed out significant variations of LDHA level, an important metabolism player, during erythroid differentiation. Deeper investigations highlighted that a metabolic switch occurred along differentiation of erythroid cells as previously emphasized in stem cell differentiation. More precisely our finding showed that self-renewing progenitor cells relied mostly upon a glycolytic, lactate-productive, metabolism and required LDHA activity, whereas differentiating cells, mainly involved the aerobic mitochondrial oxidative phosphorylation (OXPHOS). However our careful kinetic study demonstrated that these metabolic rearrangements were coming along with a particular temporary event, occurring within the first 24h of erythroid differentiation. The activity of glycolytic metabolism and OXPHOS rose jointly with ATP production at 12-24h of the differentiation process before lactate-productive glycolysis sharply fall down and energy needs decline. Finally, our results showed that the metabolic switch mediated through LDHA drop and OXPHOS upkeep might be necessary for erythroid differentiation. We also discuss the possibility that metabolism, gene expression and epigenetics could act together in a circular manner as a driving force for differentiation.

cell biology↗

bioRxiv2018

WASABI: a dynamic iterative framework for gene regulatory network inference

Inference of gene regulatory networks from gene expression data has been a long-standing and notoriously difficult task in systems biology. Recently, single-cell transcriptomic data have been massively used for gene regulatory network inference, with both successes and limitations. In the present work we propose an iterative algorithm called WASABI, dedicated to inferring a causal dynamical network from time-stamped single-cell data, which tackles some of the limitations associated with current approaches. We first introduce the concept of waves, which posits that the information provided by an external stimulus will affect genes one-by-one through a cascade, like waves spreading through a network. This concept allows us to infer the network one gene at a time, after genes have been ordered regarding their time of regulation. We then demonstrate the ability of WASABI to correctly infer small networks, which have been simulated in silico using a mechanistic model consisting of coupled piecewise-deterministic Markov processes for the proper description of gene expression at the single-cell level. We finally apply WASABI on in vitro generated data on an avian model of erythroid differentiation. The structure of the resulting gene regulatory network sheds a fascinating new light on the molecular mechanisms controlling this process. In particular, we find no evidence for hub genes and a much more distributed network structure than expected. Interestingly, we find that a majority of genes are under the direct control of the differentiation-inducing stimulus. In conclusion, WASABI is a versatile algorithm which should help biologists to fully exploit the power of time-stamped single-cell data.

systems biology↗

bioRxiv2017

Automated cell cycle and cell size measurements for single-cell gene expression studies

Recent rise of single-cell studies revealed the importance of understanding the role of cell-to-cell variability, especially at the transcriptomic level. One of the numerous sources of cell-to-cell variation in gene expression is the heterogeneity in cell proliferation state. How cell cycle and cell size influences gene expression variability at single-cell level is not yet clearly understood. To deconvolute such influences, most of the single-cell studies used dedicated methods that could include some bias. Here, we provide a universal and automatic toxic-free label method, compatible with single-cell high-throughput RT-qPCR. This led to an unbiased gene expression analysis and could be also used for improving single-cell tracking and imaging when combined with cell isolation. As an application for this technique, we showed that cell-to-cell variability in chicken erythroid progenitors was negligibly influenced by cell size nor cell cycle.

cell biology↗

bioRxiv2017

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment.

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterizing transcriptional changes in cord blood derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show, that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the two stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the two phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process, away from a simple binary switch between two options as it is usually envisioned.

cell biology↗