Search bioRxiv⌕ Search

Biology subjects

Imbert, A.

Publications and source records attributed to Imbert, A..

16 recordsLinked to original sources

BenchMHC: a reproducible framework for peptide-MHC presentation prediction

Predicting which peptides an MHC molecule presents is the first step in neoantigen and epitope discovery pipelines. Most published predictors are released for inference only, and each is trained on a different dataset, so the effect of a new method cannot be separated from the effect of its training data. We present BenchMHC, an open framework that reproduces, retrains and evaluates pMHC presentation models on public data only, and that records every implementation decision made in a reproduction. Our NetMHCpan-4.1 reproduction reaches 0.794 per-allele Mean Top-K against 0.790 for the official webserver, and 0.0025 Median FRANK against 0.0022. It scores 280,000 peptide/allele pairs in 27 s, about 350 times faster than the original binary. We then examine the training data. In the public NetMHCpan-4.1 eluted-ligand sets, 86 to 99% of hits are found in the human proteome, against about 12% of their decoys, so a model can separate hits from decoys using this difference rather than features related to MHC binding. Redrawing the decoys within the source protein of each hit and matching their cysteine status, with no other change, raises per-allele Mean Top-K on the regenerated v2.0.0 evaluation set from 0.734 to 0.755, and increases Median FRANK on CD8 epitopes from 0.0025 to 0.0053. The same change improves one evaluation set and degrades the other. Code (Apache-2.0) and model checkpoints (CC BY-NC-4.0): https://github.com/instadeepai/BenchMHC-public.

bioinformatics↗

VISIBLE: An imaging-driven system for sampling, biofabrication, and manipulation of complex biological models

Complex in vitro models (CIVMs), including organoids, spheroids, and bioprinted constructs, have emerged as powerful platforms for recapitulating human tissue architecture and function. However, their inherent heterogeneity and dynamic nature pose significant challenges for standardization, reproducibility, and real-time manipulation. Here, we present VISIBLE (Versatile Imaging-Based Interactive Sampling and Bioprinting System), a modular, imaging-guided platform that integrates real-time monitoring with automated manipulation and 3D bioprinting that addresses these challenges. VISIBLE employs a dual-axis system comprising independently controlled tool-heads and microscope stages, enabling precise spatiotemporal interventions within live cultures. We demonstrate the system capabilities across a range of applications, including morphology- and function-based sampling of organoids and neurospheres, interactive 3D bioprinting, serial scratch assays, and real-time cell-cell interaction studies. Furthermore, we illustrate the system utility in translational contexts through the selective sampling and implantation of patient-derived xenograft organoids. VISIBLE supports long-term culture within an integrated incubation environment and accommodates interchangeable tool-heads for scalable, high-throughput workflows. By enabling closed-loop, feedback-controlled experimentation, VISIBLE addresses some of the critical limitations in existing CIVM platforms and offers a new versatile solution for a wide range of biomedical research applications. This work represents a significant step toward the development of autonomous experimental systems for complex tissue modelling and preclinical investigation.

bioengineering↗

Chronobot: Deep learning guided time-resolved cryo-EM captures molecular choreography of RecA in homology search

The function of proteins and other biological macromolecules is regulated by conformational dynamics1. Many functional changes take place on millisecond timescales which cannot be experimentally captured by manual sample preparation for cryo-EM2. Here we introduce Chronobot, a robust, data-driven platform enabling reproducible, time-resolved cryo-EM sample preparation to visualize these transient intermediates. We quantify ice thickness, an important precondition and thus a reliable predictor of 3D reconstruction quality, using two complementary methods: a bespoke deep learning model analysing high-speed camera videos of the grid just before vitrification, and detailed ice thickness quantifications of outputs from common TEM screening workflows like the EPU software. Combining these methods enables rapid optimisation, resulting in an 11-fold improvement in cryo-EM sample quality compared to our previously reported workflow3. To demonstrate the Chronobot in capturing transient reaction intermediates visualised through cryo-EM and single particle analysis we focused on RecA homology search. The RecA family of recombinases perform the essential task of rapidly scanning for homologous dsDNA sequences to initiate homologous recombination. The dynamics of these RecA-dsDNA interactions occur on millisecond timescales, limiting structural insights4. We capture time-resolved homology search intermediates at 250 milliseconds. These structures reveal the involvement of the secondary DNA binding site in initial capture of dsDNA before homology sampling occurs. We also observe three-strand homology sampling intermediates, where the homologous strand is not fully displaced, and homology is not stably bound. Our results suggest a model of how RecA-family recombinases function in early homologous recombination, by coordinating the incoming DNA between RecAs various DNA binding sites depending on the stage of homology search and the presence of suUicient homology. We anticipate the Chronobot method to be broadly applicable to processes which cannot be captured by manual sample preparation methods. In addition, by leveraging AI inference, our rapid user feedback mechanisms allow for per-sample optimisation of grid conditions, increasing the likelihood of success and reducing the sample requirements of each time-resolved experiment.

biochemistry↗

PhotoNeuro: A compact photodetector for synchronization of visual stimulus presentation during behavioral experiments in neuroscience.

Presenting visual stimuli in neuroscience experiments often requires the combination of analogue signals that carry information about the visual cue presented on the LCD display. Such signals are often sensed by photodetectors and recorded in analogue to digital converter (ADC) acquisition boards. The use of open-source visual programming languages such as Bonsai is becoming more and more popular. They are often used in combination with other open-source hardware such as Arduino development boards. These microcontroller-based boards can be used to automate behavioural experiments: e.g., actuate valves and motors and acquire analogue signals on their ADC channels. LCDs and other modern display allow fast presentation of arbitrary visual stimuli and are widely used for psychophysics and neuroscience experiments. However, most displays do not provide hardware timestamping options and are intrinsically nonlinear. Solving this limitation often requires a direct recording of the light emitted by the display with a photodiode. Such photodetectors are are often amplified at higher voltages and hard to integrate in most common recording systems that use microcontrollers. The other drawback commonly found by neuroscience researchers in commercial devices is the relatively big footprint that the sensor occupies on the screen which, ideally should be minimised so not to interfere with the stimuli presentation. In this paper we present a small footprint photodetector that can be easily replicated and operates at 5V making it suitable to use with common development boards and the visual programming language Bonsai that is commonly used for experiment creation and control. Additionally, we share a version that includes four photodiodes in small area (400 mm2). Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@7bafcforg.highwire.dtl.DTLVardef@5353ccorg.highwire.dtl.DTLVardef@a29f47org.highwire.dtl.DTLVardef@86f5d2org.highwire.dtl.DTLVardef@74da5f_HPS_FORMAT_FIGEXP M_TBL C_TBL

neuroscience↗

Functional specialisation of multisensory temporal integration in the mouse superior colliculus

Our perception of the world depends on the brains ability to integrate information from multiple senses, with temporal disparities providing a critical cue for binding or segregating cross-modal signals1,2. The superior colliculus (SC) is a key site for integrating sensory modalities, but how cellular and network mechanisms in distinct anatomical regions within the SC contribute to multisensory integration remains poorly understood. Here, we recorded responses from over 5,000 neurons across the SCs anatomical axes of awake mice during presentations of spatially coincident audiovisual stimuli with varying temporal asynchronies. Our findings revealed that multisensory neurons reliably encoded audiovisual delays and exhibited nonlinear summation of auditory and visual inputs, with nonlinearities being more pronounced when visual stimuli preceded auditory stimuli, consistent with the natural statistics of light and sound propagation. Nonlinear summation was crucial for population-level decoding accuracy and precision of AV delay representation. Moreover, enhanced population decoding of audiovisual delays in the posterior-medial SC, facilitated temporal discriminability in the peripheral visual field. Cross-correlation analysis indicated higher connectivity in the medial SC and functional specific recurrent connectivity, with visual, auditory, and multisensory neurons preferentially connecting to other neurons of the same functional subclass, and multisensory neurons receiving approximately 50 percent of the total local input from other multisensory neurons. Our results highlight the interplay between single-neuron computations, network connectivity, and population coding in the SC, where nonlinear integration, distributed representations and regional functional specialisations enables robust sensory binding and supports the accurate encoding of temporal multisensory information. Our study provides new insights into how the brain leverages both single-neuron and network-level mechanisms to represent sensory features by adapting to the statistics of the natural world.

neuroscience↗

Cell cycle-dependent mRNA localization in P-bodies

Understanding the dynamics of RNA targeting to membraneless organelles is essential to disentangle their functions. Here, we investigate how P-bodies (PBs) evolve during cell cycle progression. PB purification across the cell cycle uncovers widespread changes in their RNA content, which are partly uncoupled from cell cycle-dependent changes in RNA expression. Single molecule FISH shows various mRNA localization patterns in PBs peaking in G1, S, or G2, with examples illustrating the timely capture of mRNAs in PBs when their encoded protein becomes dispensable. Yet, rather than directly reflecting absence of translation, cyclic mRNA localization in PBs can be controlled by RBPs, such as HuR in G2, and by RNA features. Indeed, while PB mRNAs are AU-rich at all cell cycle phases, they are specifically longer in G1, possibly related to post-mitotic PB reassembly. Altogether, our study supports a model where PBs are more than a default location for excess untranslated mRNAs.

cell biology↗

Transcriptomic, clonal, and functional analyses reveal Liver tissue-imprinted immuno-profile of circulating autoreactive CD4 T cells in autoimmune liver diseases

Autoimmune liver diseases (AILD) are immune-mediated disorders in which CD4 T cells play a central role. However, the link between circulating self-antigen-specific CD4 T cells and the targeted tissue has not been extensively studied in AILD. We hypothesized that circulating autoreactive CD4 T cells were clonally and functionally related to dominant intra-hepatic pathogenic CD4 T cell clones. Single cell transcriptomic analysis of circulating self-antigen-specific CD4 T cells revealed a specific B-helper and immuno-exhausted transcriptional profile, which was conserved for different autoantigens, but distinct from several other types of foreign antigen specificities. In the blood, the dominant hepatic CD4 T cell clones had a similar transcriptomic signature and were enriched in the PD-1+ TIGIT+ HLA-DR+ CD4 T cell subset. In a mouse model, antigen-specific CD4 T cells acquired the immuno-exhausted transcriptional profile when they accumulated in the liver after local antigen reactivity. Locally, immune checkpoint molecules controlled the response of antigen-specific CD4 T cells responsible for liver damage. Our study reveals the origin and biology of liver-derived autoreactive CD4 T cells in the blood of AILD patients that are imprinted by the liver environment, and suggest a dysregulation of the immune checkpoint molecules pathways. Our study enables tracking and isolating circulating autoreactive CD4 T cells for future diagnostic and therapeutic purposes.

immunology↗

Frictiotaxis underlies adhesion-independent durotaxis

Cells move directionally along gradients of substrate stiffness, a process called durotaxis. The current consensus is that durotaxis relies on cell-substrate focal adhesions to sense stiffness and transmit forces that drive directed motion. Therefore, focal adhesion-independent durotaxis is thought to be impossible. Here, we show that confined cells can perform durotaxis despite lacking strong or specific adhesions. This durotactic migration depends on asymmetric myosin distribution and actomyosin retrograde flow. We show that the mechanism of this adhesion-independent durotaxis is that stiffer substrates offer higher friction. We propose a physical model that predicts that non-adherent cells polarise and migrate towards regions of higher friction - a process that we call frictiotaxis. We demonstrate frictiotaxis in experiments by showing that cells migrate up a friction gradient even when stiffness is uniform. Our results broaden the potential of durotaxis to guide any cell that contacts a substrate and reveal a new mode of directed migration based on friction, with implications for immune and cancer cells, which commonly move with non-specific interactions.

cell biology↗

OptoPi: An open source flexible platform for the analysis of small animal behaviour

Behaviour is the ultimate output of neural circuit computations, and therefore its analysis is a cornerstone of neuroscience research. However, every animal and experimental paradigm requires different illumination conditions to capture and, in some cases, manipulate specific behavioural features. This means that researchers often develop, from scratch, their own solutions and experimental set-ups. Here, we present OptoPi, an open source, affordable ([~] {pound}600), behavioural arena with accompanying multi-animal tracking software. The system features highly customisable and reproducible visible and infrared illumination and allows for temporally precise optogenetic stimulation. OptoPi acquires images using a Raspberry Pi camera, features motorised LED-based illumination, Arduino control, as well as spectrum and irradiance monitoring to fine-tune illumination conditions with real time feedback. Our open-source software (BIO) can be used to simultaneously track multiple animals while accurately keeping individual animals identity both in on-line and off-line modes. We demonstrate the functionality of OptoPi by recording and tracking under different illumination conditions the spontaneous behaviour of larval zebrafish as well as adult Drosophila flies and their first instar larvae, an experimental animal that due to its small size and transparency has classically been hard to track. Further, we showcase OptoPis optogenetic capabilities through a series of experiments using transgenic Drosophila larvae.

neuroscience↗

A low-cost device for cryoanesthesia of neonate rodents

Studying the development of neural circuits in rodent models requires surgical access to the neonatal brain. Since commercially available stereotaxic and anesthetic equipment is designed for use in adults, reliable targeting of brain structures in such young animals can be challenging. Hypothermic cooling (cryoanesthesia) has been used as a preferred anesthesia approach in neonates. This commonly involves submerging neonates in ice, an approach that is poorly controllable. We have developed an affordable, simple to construct device - CryoPup - that allows for fast and robust cryoanesthesia of rodent pups. CryoPup consists of a microcontroller controlling a Peltier element and a heat exchanger. It is capable of both cooling and heating, thereby also functioning as a heating pad during recovery. Importantly, it has been designed for size compatibility with common stereotaxic frames. We validate CryoPup in neonatal mice, demonstrating that it allows for rapid, reliable and safe cryoanesthesia and subsequent recovery. This open-source device will facilitate future studies into the development of neural circuits in the postnatal brain. Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@de1fdorg.highwire.dtl.DTLVardef@16fde69org.highwire.dtl.DTLVardef@172f48corg.highwire.dtl.DTLVardef@15454forg.highwire.dtl.DTLVardef@79676e_HPS_FORMAT_FIGEXP M_TBL C_TBL

neuroscience↗

Combining SLA 3D printing and soft lithography for fast, versatile, and accessible high-resolution fabrication of customised multiscale cell culture devices with complex designs

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models whilst retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices and microfluidic systems to larger constructs for specific applications such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques and tools required for their manufacture, and the physical science background sometimes needed, the design and fabrication of such devices directly by biological labs remains a relatively high investment in terms of costs, use of facilities, needed collaborations and time. Whilst commercially available systems are available, these are also often costly, and importantly lack the potential for customisation by each single lab. This combination of factors still limits widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent important advancements in the fields of bioengineering and microfabrication, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised low-cost and highly reproducible microfabrication pipeline, capable of generating a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with little to none prior exposure to these fields technique and enables the manufacture of complex devices across the {micro}m to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D printed constructs for PDMS curing, using a combination of curing steps, washes and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilization of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multi-part hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.

bioengineering↗

FISH-quant v2: a scalable and modular analysis tool for smFISH image analysis

Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in-situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling a quantitative understanding of gene expression and its regulatory mechanisms. Recent progress in experimental techniques provides larger data-sets, requiring adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular analysis tool accessible both for non-experts and experts, which we validated and applied on large-scale smFISH image datasets. Our package allows the user to detect isolated and clustered mRNA spots, segment nuclei and cells, quantify RNA localization patterns and visualize these results at the single-cell level.

bioinformatics↗

The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two color imaging directly showed single mRNAs transported by single KIF1C motors, with the 3UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal an RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role both in transporting RNAs and in promoting their clustering within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA suggesting a possible feedback loop acting at the level of mRNA transport.

cell biology↗

A localization screen reveals translation factories and widespread co-translational RNA targeting

Local translation allows a spatial control of gene expression. Here, we performed a dual protein/mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. A total of 32 mRNAs displayed specific cytoplasmic localizations, and we observed local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope and centrosomes, the latter being cell cycle dependent. Quantitation of mRNA distribution and automatic pattern classification revealed a high degree of localization heterogeneity between cells. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For {beta}-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

cell biology↗

Timing and original features of flagellum assembly in trypanosomes during development in the tsetse fly

Trypanosoma brucei exhibits a complex life cycle alternating between tsetse flies and mammalian hosts. When parasites infect the fly, cells differentiate to adapt to life in various tissues, which is accompanied by drastic morphological and biochemical modifications especially in the proventriculus. This key step represents a bottleneck for salivary gland infection. Here we monitored flagellum assembly in trypanosomes during differentiation from the trypomastigote to the epimastigote stage, i.e. when the nucleus migrates to the posterior end of the cell. Three-dimensional electron microscopy (Focused Ion Bean Scanning Electron Microscopy, FIB-SEM) and immunofluorescence assays provided structural and molecular evidence that the new flagellum is assembled while the nucleus migrates towards the posterior region of the body. Two major differences with well known procyclic cells are reported. First, growth of the new flagellum begins when the associated basal body is found in a posterior position relative to the mature one. Second, the new flagellum acquires its own flagellar pocket before rotating on the left side of the anterior-posterior axis. FIB-SEM revealed the presence of a structure connecting the new and mature flagellum and serial sectioning confirmed morphological similarities with the flagella connector of procyclic cells. We discuss potential function of the flagella connector in trypanosomes from the proventriculus. These findings show that T. brucei finely modulates its cytoskeletal components to generate highly variable morphologies.\n\nAuthor SummaryTrypanosoma brucei is a flagellated parasitic protist that causes human African trypanosomiasis, or sleeping sickness and that is transmitted by the bite of tsetse flies. The complex life cycle of T. brucei inside the tsetse digestive tract requires adaptation to specific organs and follow a strictly defined order. It is marked by morphological modifications in cell shape and size, as well organelle positioning. In the proventriculus of tsetse flies, T. brucei undergoes a unique asymmetric division leading to two very different daughter cells: one with a short and one with a long flagellum. This organelle is crucial for the trypanosome life cycle as it is involved in motility, adhesion and morphogenesis. Here we investigated flagellum assembly using molecular and 3D Electron Microscopy approaches revealing that flagellum construction in proventricular trypanosomes is concomitant with parasite differentiation. During flagellum growth, the new flagellum is connected to the mature one and rotates around the mature one after its emergence at the cell surface. The sequence of events is different from what is observed in the well-studied procyclic stage in culture revealing different processes governing morphological development. These results highlight the importance to study pathogen development in their natural environment.

microbiology↗

Live Mouse Tracker: real-time behavioral analysis of groups of mice

Preclinical studies of psychiatric disorders require the use of animal models to investigate the impact of environmental factors or genetic mutations on complex traits such as decision-making and social interactions. Here, we present a real-time method for behavior analysis of mice housed in groups that couples computer vision, machine learning and Triggered-RFID identification to track and monitor animals over several days in enriched environments. The system extracts a thorough list of individual and collective behavioral traits and provides a unique phenotypic profile for each animal. On mouse models, we study the impact of mutations of genes Shank2 and Shank3 involved in autism. Characterization and integration of data from behavioral profiles of mutated female mice reveals distinctive activity levels and involvement in complex social configuration.

animal behavior and cognition↗