Acute Effects of Brevetoxin-3 Administered via Oral Gavage to Mice.

Brevetoxins (BTXs) constitute a family of lipid-soluble toxic cyclic polyethers mainly produced by Karenia brevis, which is the main vector for a foodborne syndrome known as neurotoxic shellfish poisoning (NSP) in humans. To prevent health risks associated with the consumption of contaminated shellfish in France, the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) recommended assessing the effects of BTXs via an acute oral toxicity study in rodents. Here, we investigated the effect of a single oral administration in both male and female mice with several doses of BTX-3 (100 to 1,500 µg kg-1 bw) during a 48 h observation period in order to provide toxicity data to be used as a starting point for establishing an acute oral reference dose (ARfD). We monitored biological parameters and observed symptomatology, revealing different effects of this toxin depending on the sex. Females were more sensitive than males to the impact of BTX-3 at the lowest doses on weight loss. For both males and females, BTX-3 induced a rapid, transient and dose-dependent decrease in body temperature, and a transient dose-dependent reduced muscle activity. Males were more sensitive to BTX-3 than females with more frequent observations of failures in the grip test, convulsive jaw movements, and tremors. BTX-3's impacts on symptomatology were rapid, appearing during the 2 h after administration, and were transient, disappearing 24 h after administration. The highest dose of BTX-3 administered in this study, 1,500 µg kg-1 bw, was more toxic to males, leading to the euthanasia of three out of five males only 4 h after administration. BTX-3 had no effect on water intake, and affected neither the plasma chemistry parameters nor the organs' weight. We identified potential points of departure that could be used to establish an ARfD (decrease in body weight, body temperature, and muscle activity).

One-year post-exposure assessment of 14C-few-layer graphene biodistribution in mice: single versus repeated intratracheal administration.

Research on graphene-based nanomaterials has experienced exponential growth in the last few decades, driven by their unique properties and their future potential impact on our everyday life. With the increasing production and commercialization of these materials, there is significant interest in understanding their fate in vivo. Herein, we investigated the distribution of 14C-few-layer graphene (14C-FLG) flakes (lat. dim. ∼ 500 nm) in mice over a period of one year. Furthermore, we compared the effects of repeated low-dose and acute high-dose exposure by tracheal administration. The results showed that most of the radioactivity was found in the lungs in both cases, with longer elimination times in the case of acute high-dose administration. In order to gain deeper insights into the distribution pattern, we conducted ex vivo investigations using µ-autoradiography on tissue sections, revealing the heterogeneous distribution of the material following administration. For the first time, µ-autoradiography was used to conduct a comprehensive investigation into the distribution and potential presence of FLG within lung cells isolated from the exposed lungs. The presence of radioactivity in lung cells strongly suggests internalization of the 14C-FLG particles. Overall these results show the long-term accumulation of the material in the lungs over one year, regardless of the administration protocol, and the higher biopersistence of FLG in the case of an acute exposure. These findings highlight the importance of the exposure scenario in the context of intratracheal administration, which is of interest in the evaluation of the potential health risks of graphene-based nanomaterials.

From a Cone Snail Toxin to a Competitive MC4R Antagonist.

The melanocortin 4 receptor (MC4R) plays a role in energy homeostasis and represents a target for treating energy balance disorders. For decades, synthetic ligands have been derived from MC4R endogenous agonists and antagonists, such as setmelanotide used to treat rare forms of genetic obesity. Recently, animal venoms have demonstrated their capacity to provide melanocortin ligands with toxins from a scorpion and a spider. Here, we described a cone snail toxin, N-CTX-Ltg1a, with a nanomolar affinity for hMC4R but unrelated to any known toxins or melanocortin ligands. We then derived from the conotoxin the linear peptide HT1-0, a competitive antagonist of Gs, G15, and ß-arrestin2 pathways with a low nanomolar affinity for hMC4R. Similar to endogenous ligands, HT1-0 needs hydrophobic and basic residues to bind hMC4R. Altogether, it represents the first venom-derived peptide of high affinity on MC4R and paves the way for the development of new MC4R antagonists.

C910 chemical compound inhibits the traffiking of several bacterial AB toxins with cross-protection against influenza virus.

The development of anti-infectives against a large range of AB-like toxin-producing bacteria includes the identification of compounds disrupting toxin transport through both the endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of compounds blocking Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, which was followed by orthogonal screens against two toxins that hijack the endolysosomal (diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule C910 that induces the enlargement of EEA1-positive early endosomes associated with sorting defects of CNF1 and Shiga toxins to their trafficking pathways. C910 protects cells against eight bacterial AB toxins and the CNF1-mediated pathogenic Escherichia coli invasion. Interestingly, C910 reduces influenza A H1N1 and SARS-CoV-2 viral infection in vitro. Moreover, parenteral administration of C910 to mice resulted in its accumulation in lung tissues and a reduction in lethal influenza infection.

Monitoring In Vivo Performances of Protein-Drug Conjugates Using Site-Selective Dual Radiolabeling and Ex Vivo Digital Imaging.

In preclinical models, the development and optimization of protein-drug conjugates require accurate determination of the plasma and tissue profiles of both the protein and its conjugated drug. To this aim, we developed a bioanalytical strategy based on dual radiolabeling and ex vivo digital imaging. By combining enzymatic and chemical reactions, we obtained homogeneous dual-labeled anti-MMP-14 Fabs (antigen-binding fragments) conjugated to monomethyl auristatin E where the protein scaffold was labeled with carbon-14 (14C) and the conjugated drug with tritium (3H). These antibody-drug conjugates with either a noncleavable or a cleavable linker were then evaluated in vivo. By combining liquid scintillation counting and ex vivo dual-isotope radio-imaging, it was possible not only to monitor both components simultaneously during their circulation phase but also to quantify accurately their amount accumulated within the different organs.

A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.

BACKGROUND AND PURPOSE: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action. EXPERIMENTAL APPROACH: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats. KEY RESULTS: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R. CONCLUSIONS AND IMPLICATIONS: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective.

First evidence that emerging pinnatoxin-G, a contaminant of shellfish, reaches the brain and crosses the placental barrier.

Massive proliferation of some toxic marine dinoflagellates is responsible for the occurrence of harmful algal blooms and the contamination of fish and shellfish worldwide. Pinnatoxins (PnTx) (A-H) comprise an emerging phycotoxin family belonging to the cyclic imine toxin group. Interest has been focused on these lipophilic, fast-acting and highly potent toxins because they are widely found in contaminated shellfish, and can represent a risk for seafood consumers. PnTx display a potent antagonist effect on nicotinic acetylcholine receptors (nAChR), and in this study we assessed in vivo the ability of PnTx-G to cross physiological barriers to reach its molecular target. Radiolabeled [3H]-PnTx-G synthesized with good radiochemical purity and yield retained the high affinity of the natural toxin. Oral gavage or intravenous administration to adult rats and digital autoradiographic analyses revealed the biodistribution and toxicokinetics of [3H]-PnTx-G, which is rapidly cleared from blood, and accumulates in the liver and small intestine. The labeling of peripheral and brain adult/embryo rat tissues highlights its ability to cross the intestinal, blood-brain and placental barriers. High-resolution 3D-imaging and in vitro competition studies on rat embryo sections revealed the specificity of [3H]-PnTx-G binding and its selectivity for muscle and neuronal nAChR subtypes (such as α7 subtype). The use of a human perfused cotyledon model and mass spectrometry analyses disclosed that PnTx-G crosses the human placental barrier. The increasing worldwide occurrence of both the dinoflagellate Vulcanodinium rugosum and PnTx-contaminated shellfish, due to climate warming, raises concerns about the potential adverse impact that exposure to pinnatoxins may have for human health.

AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption.

Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR).

Targeting channelrhodopsin-2 to ON-bipolar cells with vitreally administered AAV Restores ON and OFF visual responses in blind mice.

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of retinal neurons mediated by adeno-associated virus (AAV) gene therapy has the potential to restore vision regardless of patient-specific mutations. The challenge for clinical translatability is to restore a vision as close to natural vision as possible, while using a surgically safe delivery route for the fragile degenerated retina. To preserve the visual processing of the inner retina, we targeted ON bipolar cells, which are still present at late stages of disease. For safe gene delivery, we used a recently engineered AAV variant that can transduce the bipolar cells after injection into the eye's easily accessible vitreous humor. We show that AAV encoding channelrhodopsin under the ON bipolar cell-specific promoter mediates long-term gene delivery restricted to ON-bipolar cells after intravitreal administration. Channelrhodopsin expression in ON bipolar cells leads to restoration of ON and OFF responses at the retinal and cortical levels. Moreover, light-induced locomotory behavior is restored in treated blind mice. Our results support the clinical relevance of a minimally invasive AAV-mediated optogenetic therapy for visual restoration.

Spectral-domain optical coherence tomography of the rodent eye: highlighting layers of the outer retina using signal averaging and comparison with histology.

Spectral-Domain Optical Coherence Tomography (SD-OCT) is a widely used method to observe retinal layers and follow pathological events in human. Recently, this technique has been adapted for animal imaging. This non-invasive technology brings a cross-sectional visualization of the retina, which permits to observe precisely each layer. There is a clear expansion of the use of this imaging modality in rodents, thus, a precise characterization of the different outer retinal layers observed by SD-OCT is now necessary to make the most of this technology. The identification of the inner strata until the outer nuclear layer has already been clearly established, while the attribution of the layers observed by SD-OCT to the structures corresponding to photoreceptors segments and retinal pigment epithelium is much more questionable. To progress in the understanding of experimental SD-OCT imaging, we developed a method for averaging SD-OCT data to generate a mean image allowing to better delineate layers in the retina of pigmented and albino strains of mice and rats. It allowed us to locate precisely the interface between photoreceptors and retinal pigment epithelium and to identify unambiguously four layers corresponding to the inner and outer parts of photoreceptors segments. We show that the thickness of the various layers can be measured as accurately in vivo on SD-OCT images, than post-mortem by a morphometric analysis of histological sections. We applied SD-OCT to different models and demonstrated that it allows analysis of focal or diffuse retinal pathological processes such as mutation-dependent damages or light-driven modification of photoreceptors. Moreover, we report a new method of combined use of SD-OCT and integration to quantify laser-induced choroidal neovascularization. In conclusion, we clearly demonstrated that SD-OCT represents a valuable tool for imaging the rodent retina that is at least as accurate as histology, non-invasive and allows longitudinal follow-up of the same animal.