Impedance-based sensors discriminate among different types of blood thrombi with very high specificity and sensitivity.

BACKGROUND: Intracranial occlusion recanalization fails in 20% of endovascular thrombectomy procedures, and thrombus composition is likely to be an important factor. In this study, we demonstrate that the combination of electrical impedance spectroscopy (EIS) and machine learning constitutes a novel and highly accurate method for the identification of different human thrombus types. METHODS: 134 samples, subdivided into four categories, were analyzed by EIS: 29 'White', 26 'Mixed', 12 'Red' thrombi, and 67 liquid 'Blood' samples. Thrombi were generated in vitro using citrated human blood from five healthy volunteers. Histological analysis was performed to validate the thrombus categorization based on red blood cell content. A machine learning prediction model was trained on impedance data to differentiate blood samples from any type of thrombus and in between the four sample categories. RESULTS: Histological analysis confirmed the similarity between the composition of in vitro generated thrombi and retrieved human thrombi. The prediction model yielded a sensitivity/specificity of 90%/99% for distinguishing blood samples from thrombi and a global accuracy of 88% for differentiating among the four sample categories. CONCLUSIONS: Combining EIS measurements with machine learning provides a highly effective approach for discriminating among different thrombus types and liquid blood. These findings raise the possibility of developing a probe-like device (eg, a neurovascular guidewire) integrating an impedance-based sensor. This sensor, placed in the distal part of the smart device, would allow the characterization of the probed thrombus on contact. The information could help physicians identify optimal thrombectomy strategies to improve outcomes for stroke patients.

Endothelial autophagic flux hampers atherosclerotic lesion development.

Blood flowing in arteries generates shear forces at the surface of the vascular endothelium that control its anti-atherogenic properties. However, due to the architecture of the vascular tree, these shear forces are heterogeneous and atherosclerotic plaques develop preferentially in areas where shear is low or disturbed. Here we review our recent study showing that elevated shear forces stimulate endothelial autophagic flux and that inactivating the endothelial macroautophagy/autophagy pathway promotes a proinflammatory, prosenescent and proapoptotic cell phenotype despite the presence of atheroprotective shear forces. Specific deficiency in endothelial autophagy in a murine model of atherosclerosis stimulates the development of atherosclerotic lesions exclusively in areas of the vasculature that are normally resistant to atherosclerosis. Our findings demonstrate that adequate endothelial autophagic flux limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence and inflammation.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow.

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α-induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation.

Mechanical Criterion for the Rupture of a Cell Membrane under Compression.

We investigate the mechanical conditions leading to the rupture of the plasma membrane of an endothelial cell subjected to a local, compressive force. Membrane rupture is induced by tilted microindentation, a technique used to perform mechanical measurements on adherent cells. In this technique, the applied force can be deduced from the measured horizontal displacement of a microindenter's tip, as imaged with an inverted microscope and without the need for optical sensors to measure the microindenter's deflection. We show that plasma membrane rupture of endothelial cells occurs at a well-defined value of the applied compressive stress. As a point of reference, we use numerical simulations to estimate the magnitude of the compressive stresses exerted on endothelial cells during the deployment of a stent.

A simple microfluidic device to study cell-scale endothelial mechanotransduction.

Atherosclerosis is triggered by chronic inflammation of arterial endothelial cells (ECs). Because atherosclerosis develops preferentially in regions where blood flow is disturbed and where ECs have a cuboidal morphology, the interplay between EC shape and mechanotransduction events is of primary interest. In this work we present a simple microfluidic device to study relationships between cell shape and EC response to fluid shear stress. Adhesive micropatterns are used to non-invasively control EC elongation and orientation at both the monolayer and single cell levels. The micropatterned substrate is coupled to a microfluidic chamber that allows precise control of the flow field, high-resolution live-cell imaging during flow experiments, and in situ immunostaining. Using micro particle image velocimetry, we show that cells within the chamber alter the local flow field so that the shear stress on the cell surface is significantly higher than the wall shear stress in regions containing no cells. In response to flow, we observe the formation of lamellipodia in the downstream portion of the EC and cell retraction in the upstream portion. We quantify flow-induced calcium mobilization at the single cell level for cells cultured on unpatterned surfaces or on adhesive lines oriented either parallel or orthogonal to the flow. Finally, we demonstrate flow-induced intracellular calcium waves and show that the direction of propagation of these waves is determined by cell polarization rather than by the flow direction. The combined versatility and simplicity of this microfluidic device renders it very useful for studying relationships between EC shape and mechanosensitivity.

Laser induced wounding of the plasma membrane and methods to study the repair process.

Cells are constantly exposed to agents that can trigger the perforation of their plasma membrane. This damage occurs naturally, and the frequency and intensity depends on how much cells are exposed to damaging threats. The following protocol is a simple and powerful method to damage the plasma membrane using laser ablation. It allows the induction of a single and localized wound at the plasma membrane of cultured cells, which can be followed with fast time-lapse imaging. The first part of the protocol describes simple cell culture techniques and the material ideal to make the experiments. A second part of the protocol gives advice about the procedures to make effective wounds in cells while ensuring a good survival rate. We also propose different ways to follow the opening and closure of the plasma membrane. Finally, we describe the procedure to efficiently analyze the data acquired after single cell photodamage to characterize the wounding process.

ESCRT machinery is required for plasma membrane repair.

Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.

[Temporal regulation of abscission, the last step of cell division]. / Régulation temporelle de l'abscission, la dernière étape de la division cellulaire.

Cell division is one of the most tightly controlled steps of the cell cycle. Indeed, the many steps of cell division have to be perfectly coordinated both in time and space in order to ensure an error-free division and an accurate transmission of the genome from the mother cell to the two daughter cells. Abscission, the last step of cytokinesis, consists in the severing of the intercellular bridge that connects the two daughter cells after the contraction of the acto-myosin ring. As is the case for any other step of cell division, abscission has to be precisely regulated in order to take place at the right time and the proper place. Whereas the spatial regulation of abscission is quite well understood, the study of temporal regulation is in its infancy. This review begins by describing the formation of the intercellular bridge, its organization, and its composition. Next the different models of abscission are discussed. Finally, the current understanding of the temporal regulation of abscission is detailed. In particular, I present my recent results on the role of forces exerted by the daughter cells on the intercellular bridge.

ESCRT-III assembly and cytokinetic abscission are induced by tension release in the intercellular bridge.

The last step of cell division, cytokinesis, produces two daughter cells that remain connected by an intercellular bridge. This state often represents the longest stage of the division process. Severing the bridge (abscission) requires a well-described series of molecular events, but the trigger for abscission remains unknown. We found that pulling forces exerted by daughter cells on the intercellular bridge appear to regulate abscission. Counterintuitively, these forces prolonged connection, whereas a release of tension induced abscission. Tension release triggered the assembly of ESCRT-III (endosomal sorting complex required for transport-III), which was followed by membrane fission. This mechanism may allow daughter cells to remain connected until they have settled in their final locations, a process potentially important for tissue organization and morphogenesis.

Spatial control of cytokinesis by Cdr2 kinase and Mid1/anillin nuclear export.

Maintaining genome integrity and cellular function requires proper positioning of the cell division plane. In most eukaryotes, cytokinesis relies on a contractile actomyosin ring positioned by intrinsic spatial signals that are poorly defined at the molecular level. Fission yeast cells assemble a medial contractile ring in response to positive spatial cues from the nucleus at the cell center and negative spatial cues from the cell tips. These signals control the localization of the anillin-like protein Mid1, which defines the position of the division plane at the medial cortex, where it recruits contractile-ring components at mitosis onset. Here we show that Cdr2 kinase anchors Mid1 at the medial cortex during interphase through association with the Mid1 N terminus. This association underlies the negative regulation of Mid1 distribution by cell tips. We also demonstrate that the positive signaling from the nucleus is based on Mid1 nuclear export, which links division-plane position to nuclear position during early mitosis. After nuclear displacement, Mid1 nuclear export is dominant over Cdr2-dependent positioning of Mid1. We conclude that Cdr2- and nuclear export-dependent positioning of Mid1 constitute two overlapping mechanisms that relay cell polarity and nuclear positional information to ensure proper division-plane specification.

Autoprocessing of the Escherichia coli AIDA-I autotransporter: a new mechanism involving acidic residues in the junction region.

The cleavage of the autotransporter adhesin involved in diffuse adherence (AIDA-I) of Escherichia coli yields a membrane-embedded fragment, AIDAc, and an extracellular fragment, the mature AIDA-I adhesin. The latter remains noncovalently associated with AIDAc but can be released by heat treatment. In this study we determined the mechanism of AIDA-I cleavage. We showed that AIDA-I processing is an autocatalytic event by monitoring the in vitro cleavage of an uncleaved mutant protein isolated from inclusion bodies. Furthermore, by following changes in circular dichroism spectra and protease resistance of the renaturated protein, we showed that the cleavage of the protein is correlated with folding. With site-directed deletions, we showed that the catalytic activity of the protein lies in a region encompassing amino acids between Ala-667 and Thr-953, which includes the conserved junction domain of some autotransporters. With site-directed point mutations, we also found that Asp-878 and Glu-897 are involved in the processing of AIDA-I and that a mutation preserving the acidic side chain of Asp-878 was tolerated, giving evidence that this carboxylic acid group is directly involved in catalysis. Last, we confirmed that cleavage of AIDA-I is intramolecular. Our results unveil a new mechanism of auto-processing in the autotransporter family.