Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to ...control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (Na
) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal Na
channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.
The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp ...technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.
Understanding the role of microbial interactions in the functioning of natural systems is often impaired by the levels of complexity they encompass. In this study, we used the relative simplicity of ...an hypersaline crater lake hosting only microbial organisms (Dziani Dzaha) to provide a detailed analysis of the microbial networks including the three domains of life. We identified two main ecological zones, one euphotic and oxic zone in surface, where two phytoplanktonic organisms produce a very high biomass, and one aphotic and anoxic deeper zone, where this biomass slowly sinks and undergoes anaerobic degradation. We highlighted strong differences in the structure of microbial communities from the two zones and between the microbial consortia associated with the two primary producers. Primary producers sedimentation was associated with a major reorganization of the microbial network at several levels: global properties, modules composition, nodes and links characteristics. We evidenced the potential dependency of Woesearchaeota to the primary producers' exudates in the surface zone, and their disappearance in the deeper anoxic zone, along with the restructuration of the networks in the anoxic zone toward the decomposition of the organic matter. Altogether, we provided an in-depth analysis of microbial association network and highlighted putative changes in microbial interactions supporting the functioning of the two ecological zones in this unique ecosystem. Keywords: archaea; bacteria; picoeukaryota; hypersaline ecosystem; microbial association network; Arthrospira/usiformis; Picocystis salinarum
Thalassohaline ecosystems are hypersaline environments originating from seawater in which sodium chloride is the most abundant salt and the pH is alkaline. Studies focusing on microbial diversity in ...thalassohaline lakes are still scarce compared with those on athalassohaline lakes such as soda lakes that have no marine origin. In this work, we investigated multiple facets of bacterial, archaeal and eukaryotic diversity in the thalassohaline Lake Dziani Dzaha using a metabarcoding approach. We showed that bacterial and archaeal diversity were mainly affected by contrasting physicochemical conditions retrieved at different depths. While photosynthetic microorganisms were dominant in surface layers, chemotrophic phyla (Firmicutes or Bacteroidetes) and archaeal methanogens dominated deeper layers. In contrast, eukaryotic diversity was constant regardless of depth and was affected by seasonality. A detailed focus on eukaryotic communities showed that this constant diversity profile was the consequence of the high predominance of Picocystis salinarum, while nondominant eukaryotic groups displayed seasonal diversity turnover. Altogether, our results provided an extensive description of the diversity of the three domains of life in an unexplored extreme environment and showed clear differences in the responses of prokaryotic and eukaryotic communities to environmental conditions.
Japanese Knotweeds were introduced by man during the 19
th
century in Europe, where they hybridized and formed a species complex now considered as "invasive". On the basis of many herbarium ...specimens, the literature and archive documents, we describe the history of their discovery and introduction in France. The circumstances of their hybridization are given, as well as the reasons for the spread of the complex composed of both taxa and their polyploid hybrids. We show that after its discovery by Von Siebold, Reynoutria japonica was introduced as a single male-sterile clone in Lorraine during the early 1850s, whereas R. sachalinensis was discovered by both the French and Russians and subject to successive introductions. Our study reveals that at the end of the 19
th
century, these Asiatic Knotweeds became very popular among horticulturists and botanical gardens. Their cultivation in sympatry has led to the production of their hybrid R. x bohemica, as well as interspecific hybridizations and introgressions with parental species, leading to considerable genetic diversity, escape and naturalisation.
I
current, a major component of cardiac repolarization, is mediated by human
-Related Gene (hERG, K
11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated ...to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.
The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that ...poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.
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The scorpion toxin AmmTx3 is a specific blocker of Kv4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical ...synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαβ structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.
The composition of the juice from grape berries is at the basis of the definition of technological ripeness before harvest, historically evaluated from global sugar and acid contents. If many studies ...have contributed to the identification of other primary and secondary metabolites in whole berries, deepening knowledge about the chemical composition of the sole flesh of grape berries (i.e., without considering skins and seeds) at harvest is of primary interest when studying the enological potential of widespread grape varieties producing high-added-value wines. Here, we used non-targeted DI-FT-ICR-MS and RP-UHPLC-Q-ToF-MS analyses to explore the extent of metabolite coverage of up to 290 grape juices from four
grape varieties, namely Chardonnay, Pinot noir, Meunier, and Aligoté, sampled at harvest from 91 vineyards in Europe and Argentina, over three successive vintages. SPE pretreatment of samples led to the identification of more than 4500 detected C,H,O,N,S-containing elemental compositions, likely associated with tens of thousands of distinct metabolites. We further revealed that a major part of this chemical diversity appears to be common to the different juices, as exemplified by Pinot noir and Chardonnay samples. However, it was possible to build significant models for the discrimination of Chardonnay from Pinot noir grape juices, and of Chardonnay from Aligoté grape juices, regardless of the geographical origin or the vintage. Therefore, this metabolomic approach opens access to a remarkable holistic molecular description of the instantaneous composition of such a biological matrix, which is the result of complex interplays among environmental, biochemical, and vine growing practices.
Phlotoxin-1 (PhlTx1) is a peptide previously identified in tarantula venom (
species) that belongs to the inhibitory cysteine-knot (ICK) toxin family. Like many ICK-based spider toxins, the synthesis ...of PhlTx1 appears particularly challenging, mostly for obtaining appropriate folding and concomitant suitable disulfide bridge formation. Herein, we describe a procedure for the chemical synthesis and the directed sequential disulfide bridge formation of PhlTx1 that allows for a straightforward production of this challenging peptide. We also performed extensive functional testing of PhlTx1 on 31 ion channel types and identified the voltage-gated sodium (Na
) channel Na
1.7 as the main target of this toxin. Moreover, we compared PhlTx1 activity to 10 other spider toxin activities on an automated patch-clamp system with Chinese Hamster Ovary (CHO) cells expressing human Na
1.7. Performing these analyses in reproducible conditions allowed for classification according to the potency of the best natural Na
1.7 peptide blockers. Finally, subsequent in vivo testing revealed that intrathecal injection of PhlTx1 reduces the response of mice to formalin in both the acute pain and inflammation phase without signs of neurotoxicity. PhlTx1 is thus an interesting toxin to investigate Na
1.7 involvement in cellular excitability and pain.
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