Biology subjects

Brun, S.

Publications and source records attributed to Brun, S..

9 recordsLinked to original sources

bioRxiv2026

Hydrogel confinement enables subcutaneous delivery of vesicant antibody-drug conjugates

Antibody-drug conjugates (ADCs) deliver cytotoxic payloads to tumors with antibody selectivity, yet all approved ADCs are administered by intravenous (IV) infusion despite a strong patient and clinical preference for subcutaneous (SC) delivery. SC administration would reduce treatment burden, but many ADC payloads are vesicants that cause tissue necrosis upon local release, a liability amplified, not mitigated, by the dispersion-enhancing excipients used for SC antibody formulations. We developed an injectable diacetyl-L-tartaric anhydride-functionalized chitosan hydrogel (TACT) that addresses this conflict by confining ADCs within a protective SC depot. TACT is compatible with clinically approved ADC formulations without drug-product modification and provides drug-to-antibody ratio (DAR)-dependent release kinetics that support a quantitative relationship with in vivo absorption timing. In direct comparison, recombinant human hyaluronidase (rHuPH20) co-formulated with vesicant ADCs caused severe tissue necrosis, whereas TACT prevented macroscopic injury while preserving antitumor efficacy comparable to intravenous dosing. TUNEL staining of injection sites showed that TACT attenuated peri-depot apoptotic injury 3-fold relative to T-DM1 alone and 2-fold relative to rHuPH20 co-formulation. In non-human primates, SC TACT achieved 78% relative bioavailability for total trastuzumab, reduced peak circulating T-DM1 catabolite (free DM1) exposure 7.6-fold compared to IV administration and produced only transient, self-resolving cutaneous reactions. These results identify depot-mediated confinement as a viable alternative to excipient-mediated dispersion for SC delivery of vesicant ADCs, demonstrated here for trastuzumab-based conjugates across two approved ADC drug products (T-DM1 and T-DXd, with non-cleavable MCC and cleavable peptide linkers), with supporting validation in a custom cleavable monomethyl auristatin E (MMAE) series. Additional validation with enfortumab vedotin (EV), a Nectin-4-targeting MMAE ADC, supported the applicability of this strategy beyond trastuzumab-based conjugates.

cancer biology↗

bioRxiv2025

The human factor: development and characterization of a scalable calcium imaging assay using human iPSC-derived neurons

Neuroscience drug discovery is challenged by the brains structural and cell-type complexity, which is difficult to model in cellular systems compatible with high-throughput screening methods. Calcium oscillation assays, that harness neurons intrinsic capability to develop functional neural networks in cell culture, are currently the closest cellular models with a relevant functional endpoint to model human neuronal circuitry in a dish. Here we further developed this useful assay towards scalable drug discovery applications. We show the importance of defined neuron-to-astrocyte ratios for optimal cellular distribution and surface adherence in HTS-compatible cell culture vessels and how the cell type ratios affect network firing patterns. Increasing the neuron density resulted in decreased network spike frequencies, but increased network spike amplitudes. We identified DAPT, a molecule previously shown to promote neuronal maturation and synapse formation, as a negative regulator of astrocyte viability. Furthermore, inclusion of GABAergic neurons in the cocultures increased the network spike frequency while reducing network spike amplitudes. The GABA A receptor antagonist bicuculline did not affect network spike frequency, but increased network spike amplitudes. In order to access local field activity in an automated and scalable calcium imaging environment, we developed a pixel-based analysis for plate reader data. This method revealed that the effect of GABAergic neurons and bicuculline was restricted to local field calcium activity that coincided with synchronized network spikes. Our observations are consistent with previous findings suggesting that the presence of GABAergic neurons decreases synchronization and network spike participation of local neuronal activity, thus potentially echoing aspects of GABA action in vivo, and dysregulation thereof in pathological conditions.

neuroscience↗

bioRxiv2025

The fastest movements and the most extreme deformations of the cell nucleus are driven by microtrubules in the model fungus Podospora anserina

A growing number of studies shows that nuclear movement, positioning, and the mechanical constraints applied to the nucleus play important roles in cellular physiology and gene regulation. This process, known as nuclear mechanotransduction, is an emerging field of research, and its deregulation can lead to severe human pathologies. In this study, we examine the behavior of female and male nuclei during sexual reproduction in the model fungus Podospora anserina. After plasmogamy, the two types of nuclei do not immediately undergo karyogamy. Instead, once inside the trichogyne (the female-specific hyphae extending from the ascogonium) the male nuclei migrate over distances of up to several hundred micrometers from the trichogyne tip, where plasmogamy occurs, to the ascogonium at the center of the protoperithecium. Our study highlights the contrasting behaviors of the two nuclear types: female nuclei remain immobile and spherical, while male nuclei move rapidly and exhibit striking deformations and stretching. Trichogynes are branched and compartmentalized, consisting of articles separated by septa that communicate through the septal pores. We show that during their migration across these compartments, male nuclei undergo the most extreme movements, deformations, and speeds reported to date for nuclei in motion. We also investigated the role of the actin and microtubule (MT) cytoskeletons in male nuclei behavior. Both cytoskeletal networks are highly dynamic and abundant in trichogynes, particularly at septa. We demonstrate that while actin depolymerization does not impair male nuclei migration, MT disruption causes a complete arrest of both nuclear movement and stretching, indicating a critical role for MTs. Finally, we reveal that the MT network in trichogynes is highly polarized, offering new insights into how male nuclei navigate within branched trichogynes.

cell biology↗

bioRxiv2024

Prevalence of heritable symbionts in the Parisian Bedbugs (Hemiptera: Cimicidae)

Like many insects, the biology of bedbugs is impacted by a range of partner heritable microbes. Three maternally inherited symbionts are recognised, Wolbachia (an obligate partner), Symbiopectobacterium purcelli strain SyClec and Ca. Tisiphia sp. (facultative symbionts typically present in some but not all individuals). Past work had examined these heritable microbes from established laboratory lines, but not from broader field samples. We therefore deployed targeted endpoint PCR assays to determine the symbiont infection status for 50 bedbugs collected from 10 districts of Paris during the 2023 outbreak. All three symbionts were found to be broadly present across C. lectularius samples with the Symbiopectobacterium- Ca. Tisiphia-Wolbachia triple infection most commonly observed. A minority of individuals lacked either one or both facultative symbionts. Five mtDNA haplotypes were observed across the COI barcode region. Triple infections were found in all mtDNA haplotypes, indicating symbiont infection had been circulating over a protracted period of time. We conclude that the Parisian bedbug outbreak was one in which the hosts secondary symbionts were present at high frequency coinfections. Thus, facultative symbionts are an important but uncharacterized component of bedbug populations.

microbiology↗

bioRxiv2022

The GUN mutants: new weapons to unravel ascospore germination regulation in the model fungus Podospora anserina

In Podospora anserina as in many other ascomycetes, ascospore germination is a regulated process that requires breaking of dormancy. Despite its importance in survival and dispersal, ascospore germination in filamentous fungi has been poorly investigated and little is known about its regulation and genetic control. We have designed a positive genetic screen that led to the isolation of mutants showing uncontrolled germination, the GUN mutants. In this paper, we report on the characterization of GUN1SG. We show that GUN1SG is mutated in Pa_6_1340, the ortholog of Magnaporthe oryzae Pth2, which encodes a Carnitine-acetyltransferase (CAT) involved in the shuttling of acetyl-CoA between peroxisomes and mitochondria and which is required for appressorium-development. Bioinformatic analysis revealed that the mutated residue (I441) is highly conserved among the Fungi, and that the mutation has a deleterious impact on the protein function. We show that GUN1 is essential for ascospore germination and that the protein is localized both in mitochondria and in peroxisomes. Finally, epistasis studies allowed us to place GUN1 upstream of the PaMpk2 MAPK pathway and the PaNox2/PaPls1 complex in the regulation of ascospore germination. The identification of GUN1, the ortholog of Pth2, in ascospore germination, strengthens the idea of a common genetic regulation governing both appressorium development and melanized ascospore germination. In addition, we characterize the second CAT encoded in P. anserina genome, Pa_3_7660/GUP1, and we show that the function of both CATs is conserved in P. anserina.

genetics↗

bioRxiv2021

Courtship ritual of male and female nuclei during fertilisation in Neurospora crassa

Sexual reproduction is a key process influencing the evolution and adaptation of animals, plants and many eukaryotic microorganisms, such as fungi. Mycologists have described the different fungal fruiting bodies, while geneticists have partly unravelled the regulation of sexual development. However, the sequential fungal cell biology of fertilisation and the associated nuclear dynamics after plasmogamy are poorly understood. Using histone-fluorescent parental isolates, we tracked male and female nuclei during fertilisation in the model ascomycetes Neurospora crassa using live-cell-imaging. This study unravels the behaviour of trichogyne resident female nuclei and the extraordinary manner that male nuclei migrate up the trichogyne to the protoperithecium. Our observations raise new fundamental questions about the modus operandi of nuclei movements during sexual reproduction, male and female nuclear identity, guidance of nuclei within the trichogyne and, unexpectedly, the avoidance of "polyspermy" in fungi. The spatio-temporal dynamics of male nuclei within the trichogyne following plasmogamy are also described, where the speed and the deformation of male nuclei are of the most dramatic observed to date in a living organism.

cell biology↗

bioRxiv2020

GNS561, a new autophagy inhibitor active against cancer stem cells in hepatocellular carcinoma and hepatic metastasis from colorectal cancer

Patients with advanced hepatocellular carcinoma (HCC) or metastatic colorectal cancer (mCRC) have a very poor prognosis due to the lack of efficient treatments. As observed in several other tumors, the effectiveness of treatments is mainly hampered by the presence of a highly tumorigenic sub-population of cancer cells called cancer stem cells (CSCs). Indeed, CSCs are resistant to chemotherapy and radiotherapy and have the ability to regenerate the tumor bulk. Hence, innovative drugs that are efficient against both bulk tumor cells and CSCs would likely improve cancer treatment. In this study, we demonstrated that GNS561, a new autophagy inhibitor that induces lysosomal cell death, showed significant activity against not only the whole tumor population but also a sub-population displaying CSC features (high ALDH activity and tumorsphere formation ability) in HCC and in liver mCRC cell lines. These results were confirmed in vivo in HCC from a DEN-induced cirrhotic rat model in which GNS561 decreased tumor growth and reduced the frequency of CSCs (CD90+CD45-). Accordingly, GNS561, which was in a global phase 1b clinical trial in liver cancers that was recently successful, offers great promise for cancer therapy by exterminating both the tumor bulk and the CSC sub-population.

cancer biology↗

bioRxiv2020

Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France

Through routine genomic surveillance of the novel SARS-CoV-2 virus (n=229 whole genome sequences), 2 different frameshifting deletions were newly detected in the open reading frame (ORF) 6, starting at the same position (27267). While the 26-nucleotide deletion variant was only found in one sample in March 2020, the 34-nucleotide deletion variant was found within a single geriatric hospital unit in 5/9 patients sequenced and one health care worker with samples collected between April 2nd and 9th, 2020. Both the presence of the 34-nucleotide deletion variant limited to this unit and the clustering of the corresponding whole genome sequences by phylogeny analysis strongly suggested a nosocomial transmission between patients. Interestingly, prolonged viral excretion of the 34-nucleotide deletion variant was identified in a stool sample 14 days after initial diagnosis for one patient. Clinical data revealed no significant difference in disease severity between patients harboring the wild-type or the 34-nucleotide deletion variants. The in vitro infection of the two deletion variants on primate endothelial kidney cells (BGM) and human lung adenocarcinoma cells (Calu-3) yielded comparable replication kinetics with the wild-type strain. Furthermore, high viral loads were found in vivo regardless of the presence or absence of the ORF6 deletion. Our study highlights the transmission and replication capacity of two newly described deletion variants in the same ORF6 region. ImportanceWhile the SARS-CoV-2 genome has remained relatively stable since its emergence in the human population, genomic deletions are an evolutionary pattern previously described for the related SARS-CoV. Real-time genomic monitoring of the circulating variants is paramount to detect strain prevalence and transmission dynamics. Given the role of ORF6 in interferon modulation, further characterization, such as mechanistic interactions and interferon monitoring in patients, is crucial in understanding the viral-host factors driving disease evolution.

microbiology↗

bioRxiv2020

Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation

Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct [≥]25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.

genomics↗