Search

PIEZO1 is a mechanosensitive non-selective cation channel, present in many cell types including Red Blood Cells (RBCs). Together with the Gárdos channel, PIEZO1 forms in RBCs a tandem that participates in the rapid adjustment of the cell volume. The pharmacology allowing functional studies of the roles of PIEZO1 has only recently been developed, with Yoda1 as a widely used PIEZO1 agonist. In 2018, Yoda1 analogues were developed, as a step towards an improved understanding of PIEZO1 roles and functions. Among these, Dooku1 was the most promising antagonist of Yoda1-induced effects, without having any ability to activate PIEZO1 channels. Since then, Dooku1 has been used in various cell types to antagonize Yoda1 effects. In the present study using RBCs, Dooku1 shows an apparent IC 50 on Yoda1 effects of 90.7 µM, one order of magnitude above the previously reported data on other cell types. Unexpectedly, it was able, by itself , to produce entry of calcium sufficient to trigger Gárdos channel activation. Moreover, Dooku1 evoked a rise in intracellular sodium concentrations, suggesting that it targets a non-selective cation channel. Dooku1 effects were abolished upon using GsMTx4, a known mechanosensitive channel blocker, indicating that Dooku1 likely targets PIEZO1. Our observations lead to the conclusion that Dooku1 behaves as a PIEZO1 agonist in the RBC membrane, similarly to Yoda1 but with a lower potency. Taken together, these results show that the pharmacology of PIEZO1 in RBCs must be interpreted with care especially due to the unique characteristics of RBC membrane and associated cytoskeleton., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hatem, Poussereau, Gachenot, Pérès, Bouyer and Egée.)

Introduction: In paediatric rehabilitation, fun and motivation are also critical keys to successful therapy. A variety of interventions have shown positive effects, high level of interest, compliance and engagement with active video game (AVG).This seems to be an interesting approach for the postoperative gait rehabilitation of children with cerebral palsy (CP). In this study, we will investigate if an overground gait training (GT) delivered through an AVG can improve walking capacity and anaerobic performance., Methods and Analysis: This study is a randomised clinical controlled trial. A total of 14 children and adolescents in the age of 10-18 years with CP will be included. The minimum time between surgery and inclusion will be 7 weeks. The test group will participate in the GT programme with Augmented Reality Rehabilitation of Walking-Cerebral Palsy AVG, control group will receive GT on a treadmill. The primary outcome is the 6-Min Walk Test assessing walking capacity; secondary outcomes are the Muscle Power Sprint Test for anaerobic performance and Shuttle Run Test for physical fitness level. Satisfaction is tested with the Physical Activity Enjoyment Scale., Ethics and Dissemination: The findings will be disseminated by publications in peer-reviewed journals and conferences. This study received agreement from French ethic committee (Comité de Protection des Personnes Sud-Est VI-Number 2020-A02959-30)., Trial Registration Number: NCT04837105., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In an augmented reality environment, the range of possible real-time visual feedback is extensive. This study aimed to compare the impact of six scenarios in augmented reality combining four visual feedback characteristics on achieving a target walking speed. The six scenarios have been developed for Microsoft Hololens augmented reality headset. The four feedback characteristics that we have varied were: Color; Spatial anchoring; Speed of the feedback, and Persistence. Each characteristic could have different values (for example, the color could be unicolor, bicolor, or gradient). Participants had to walk for two consecutive walking trials for each scenario: at their maximal speed and an intermediate speed. Mean speed, percentage of time spent above or around target speed, and time to reach target speed were compared between scenarios using mixed linear models. A total of 25 children with disabilities have been included. The feasibility and user experience were excellent. Mean speed during scenario 6, which displayed feedback with gradient color, attached to the world, with a speed relative to the player equal to his speed, and that disappeared over time, was significantly higher than other scenarios and control (p =0.003). Participants spent 80.98% of time above target speed during scenario 6. This scenario mixed the best combination of feedback characteristics to exceed the target walking speed (p=0.0058). Scenarios 5 and 6, which shared the same feedback characteristics for spatial anchoring (world-locked) and feedback speed (equal to the player speed), decreased the time to reach the target speed (p=0.019). Delivering multi-modal feedback has been recognized as more effective for improving motor performance. Therefore, our results showed that not all visual feedback had the same impact on performance. Further studies are required to test the weight of each feedback characteristic and their possible interactions inside each scenario. This study was registered in the ClinicalTrials.gov database (NCT04460833).

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed P cat . Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Monedero Alonso, Pérès, Hatem, Bouyer and Egée.)

Serious games are a promising approach to improve gait rehabilitation for people with gait disorders. Combined with wearable augmented reality headset, serious games for gait rehabilitation in a clinical setting can be envisaged, allowing to evolve in a real environment and provide fun and feedback to enhance patient's motivation. This requires a method to obtain accurate information on the spatiotemporal gait parameters of the playing patient. To this end, we propose a new algorithm called HoloStep that computes spatiotemporal gait parameters using only the head pose provided by an augmented reality headset (Hololens). It is based on the detection of peaks associated to initial contact event, and uses a combination of locking distance, locking time, peak amplitude detection with custom thresholds for children with CP. The performance of HoloStep was compared during a walking session at comfortable speed to Zeni's reference algorithm, which is based on kinematics and a full 3D motion capture system. Our study included 62 children with cerebral palsy (CP), classified according to Gross Motor Function Classification System (GMFCS) between levels I and III, and 13 healthy participants (HP). Metrics such as sensitivity, specificity, accuracy and precision for step detection with HoloStep were above 96%. The Intra-Class Coefficient between steps length calculated with HoloStep and the reference was 0.92 (GMFCS I), 0.86 (GMFCS II/III) and 0.78 (HP). HoloStep demonstrated good performance when applied to a wide range of gait patterns, including children with CP using walking aids. Findings provide important insights for future gait intervention using augmented reality games for children with CP.

To ensure the transport of nutrients necessary for their survival, Plasmodium falciparum parasites increase erythrocyte permeability to diverse solutes. These new permeation pathways (NPPs) have been extensively characterized in the pathogenic asexual parasite stages, however the existence of NPPs has never been investigated in gametocytes, the sexual stages responsible for transmission to mosquitoes. Here, we show that NPPs are still active in erythrocytes infected with immature gametocytes and that this activity declines along gametocyte maturation. Our results indicate that NPPs are regulated by cyclic AMP (cAMP) signaling cascade, and that the decrease in cAMP levels in mature stages results in a slowdown of NPP activity. We also show that NPPs facilitate the uptake of artemisinin derivatives and that phosphodiesterase (PDE) inhibitors can reactivate NPPs and increase drug uptake in mature gametocytes. These processes are predicted to play a key role in P. falciparum gametocyte biology and susceptibility to antimalarials.

Two main isoforms of the Translocator Protein (TSPO) have been identified. TSPO1 is ubiquitous and is mainly present at the outer mitochondrial membrane of most eukaryotic cells, whereas, TSPO2 is specific to the erythroid lineage, located at the plasma membrane, the nucleus, and the endoplasmic reticulum. The design of specific tools is necessary to determine the molecular associations and functions of TSPO, which remain controversial nowadays. We recently demonstrated that TSPO2 is involved in a supramolecular complex of the erythrocyte membrane, where micromolar doses of the classical TSPO ligands induce ATP release and zinc protoporphyrin (ZnPPIX) transport. In this work, three newly-designed ligands (NCS1016, NCS1018, and NCS1026) were assessed for their ability to modulate the functions of various erythrocyte's and compare them to the TSPO classical ligands. The three new ligands were effective in reducing intraerythrocytic Plasmodium growth, without compromising erythrocyte survival. While NCS1016 and NCS1018 were the most effective ligands in delaying sorbitol-induced hemolysis, NCS1016 induced the highest uptake of ZnPPIX and NCS1026 was the only ligand inhibiting the cholesterol uptake. Differential effects of ligands are probably due, not only, to ligand features, but also to the dynamic interaction of TSPO with various partners at the cell membrane. Further studies are necessary to fully understand the mechanisms of the TSPO's complex activation.

The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Iron is an essential micronutrient but is also highly toxic. In yeast and plant cells, a key detoxifying mechanism involves iron sequestration into intracellular storage compartments, mediated by members of the vacuolar iron-transporter (VIT) family of proteins. Here we study the VIT homologue from the malaria parasites Plasmodium falciparum (PfVIT) and Plasmodium berghei (PbVIT). PfVIT-mediated iron transport in a yeast heterologous expression system is saturable (Km ∼ 14.7 μM), and selective for Fe(2+) over other divalent cations. PbVIT-deficient P. berghei lines (Pbvit(-)) show a reduction in parasite load in both liver and blood stages of infection in mice. Moreover, Pbvit(-) parasites have higher levels of labile iron in blood stages and are more sensitive to increased iron levels in liver stages, when compared with wild-type parasites. Our data are consistent with Plasmodium VITs playing a major role in iron detoxification and, thus, normal development of malaria parasites in their mammalian host.

Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature.

Plasmodium falciparum relies on anion channels activated in the erythrocyte membrane to ensure the transport of nutrients and waste products necessary for its replication and survival after invasion. The molecular identity of these anion channels, termed "new permeability pathways" is unknown, but their currents correspond to up-regulation of endogenous channels displaying complex gating and kinetics similar to those of ligand-gated channels. This report demonstrates that a peripheral-type benzodiazepine receptor, including the voltage dependent anion channel, is present in the human erythrocyte membrane. This receptor mediates the maxi-anion currents previously described in the erythrocyte membrane. Ligands that block this peripheral-type benzodiazepine receptor reduce membrane transport and conductance in P falciparum-infected erythrocytes. These ligands also inhibit in vitro intraerythrocytic growth of P falciparum. These data support the hypothesis that dormant peripheral-type benzodiazepine receptors become the "new permeability pathways" in infected erythrocytes after up-regulation by P falciparum. These channels are obvious targets for selective inhibition in anti-malarial therapies, as well as potential routes for drug delivery in pharmacologic applications.

During the past three decades, electrophysiological studies revealed that human red blood cell membrane is endowed with a large variety of ion channels. The physiological role of these channels, if any, remains unclear; they do not participate in red cell homeostasis which is rather based on the almost total absence of cationic permeability and minute anionic conductance. They seem to be inactive in the "resting cell." However, when activated experimentally, ion channels can lead to a very high single cell conductance and potentially induce disorders, with the major risks of fast dehydration and dissipation of gradients. Could there be physiological conditions under which the red cell needs to activate these high conductances, or are ion channels relics of a function lost in anucleated cells? It has been demonstrated that they play a key role in diseases such as sickle cell anemia or malaria. This short overview of ion channels identified to-date in the human red cell membrane is an attempt to propose a dynamic role for these channels in circulating cells in health and disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Historically, the anion transport through the human red cell membrane has been perceived to be mediated by Band 3, in the two-component concept with the large electroneutral anion exchange accompanied by the conductance proper, which dominated the total membrane conductance. The status of anion channels proper has never been clarified, and the informations obtained by different groups of electrophysiologists are rather badly matched. This study, using the cell-attached configuration of the patch-clamp technique, rationalizes and explains earlier confusing results by demonstrating that the diversity of anionic channel activities recorded in human erythrocytes corresponds to different kinetic modalities of a unique type of maxi-anion channel with multiple conductance levels and probably multiple gating properties and pharmacology, depending on conditions. It demonstrates the role of activator played by serum in the recruitment of multiple new conductance levels showing very complex kinetics and gating properties upon serum addition. These channels, which seem to be dormant under normal physiological conditions, are potentially activable and could confer a far higher anion conductance to the red cell than the ground leak mediated by Band 3.

Background: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents., Methodology/principal Findings: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents., Conclusions/significance: The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia.