Ethylene-propylene-diene monomer rubber, grafted with maleic anhydride functions (EPDM-g-MA) was organically reinforced by reactive extrusion from the in situ synthesis of a polyimide (PI) phase. The blends were reactively processed at 200 degrees C in a twin-screw extruder, with the PI content varying from 5 to 40 wt%. Transmission Electron Microscopy (TEM) revealed a very fine nodular dispersion of the PI phase with diameters ranging from 130 to 240 nm depending on the PI concentration. The reinforcement of the elastomer was evidenced with a sharp increase of the Young's modulus and the tensile strength, and the stiffness of the material was further increased with the PI content. The linear viscoelastic regime, as measured by the variation in storage modulus as a function of strain, was unaffected by this organic reinforcement, thus opening up an original way of controlling the Payne effect. Additionally, the cross-linking of the blend with 20 wt% of PI with dicumyl peroxide (DCP) showed that the PI phase did not impact the creation of the cross-linked network. The decrease of the swelling ratio and the improvement of the elastic recovery suggested that EPDM-g-PI copolymers can create a second network in the material, resulting in a higher apparent cross-linking density. The mechanical properties of the cured blend showed a doubling of the Young's modulus and maximal stress values compared to those obtained for the pure matrix as well as a constant strain at break.Highlights Ethylene-propylene-diene monomer rubber, grafted with maleic anhydride functions (EPDM-g-MA) was organically reinforced by reactive extrusion. The reinforcement was obtained from the in situ synthesis of a polyimide (PI) phase. The Young's modulus of cross-linked EPDM containing 20 wt% PI was doubled. The linear viscoelastic regime was unaffected by this organic reinforcement. This original way of reinforcement opens the control of the Payne effect. Mechanical properties of crosslinked EPDM reinforced with a polyimide dispersed phase. image

Selective and targeted sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors represent a promising and new class of cancer chemotherapeutics that are under investigation in different clinical trials. However, resistance to cancer chemotherapeutics treatments is a common phenomenon and recent evidence suggests that SERCA inhibition could also lead to innate and/or acquired ability of cancer cells to evade cell death but the molecular mechanisms are not fully elucidated. In LNCaP prostate cancer cells, we discovered that activation of the PERK branch of the unfolded protein response (UPR) pathways is involved in therapeutic escape of the endoplasmic reticulum (ER)-Ca2+ stress inducer ASP-8ADT, the active compound of Mispsagargin. We show that the activation of the Akt pathway in response to UPR allows the survival of LNCaP cells to ASP- 8ADT exposure. Interestingly, pharmacological inhibition of the PERK downstream factors GSK-3β or eIF2α by drugs currently used in clinic greatly sensitizes LNCaP cells to ASP-8ADT. In summary, our findings highlight additional strategies to increase clinical response to targeting-ERCa2+ stressor drugs such as Mipsagargin family.

Polypropylene/polyimide (PP/PI) blends were synthesized by reactive extrusion with the in situ polymerization of the polyimide phase in presence of maleic anhydride grafted polypropylene (PP-g-MA) as a compatibilizer. Blends were reactively processed in a twin-screw extruder at 200 ?, with the PI content varying from 10 to 40 wt %. Examination of morphology by scanning electron microscopy (SEM) depicted a fine sub-micrometer dispersion of the polyimide phase ranging from 170 to 250 nm in diameter, for all PI concentrations. The advantage of the in situ approach compared to the more classical dispersion process was clearly demonstrated. The in situ synthesized PI phase was characterized by NMR and FTIR after separation by Soxhlet selective extraction. Differential scanning calorimetry (DSC) analysis of the blends showed no modifications of PP crystallinity with the addition of PI. Rheological behaviors showed a significant impact of the polyimide phase on the storage modulus with the appearance of a secondary storage modulus plateau at low frequencies. Thermogravimetric analyses evidenced an improvement of the thermal stability of the blends, with an increase of the decomposition temperature up to 35 ?depending on the polyimide content. Mechanical properties were also improved due to the high mechanical strength of the polyimide phase, with an increase in Young's modulus up to +35 % for the highest polyimide content.

The mammalian exclusive Orai3 channel participates in the generation and/or modulation of two independent Ca2+ currents, the store-operated current, Icrac, involving functional interactions between the stromal interaction molecules (STIM), STIM1/STIM2, and Orai1/Orai2/Orai3, as well as the store-independent arachidonic acid (AA) (or leukotriene C4)-regulated current Iarc, which involves Orai1, Orai3 and STIM1. Overexpression of functional Orai3 has been described in different neoplastic cells and cancer tissue samples as compared to non-tumor cells or normal adjacent tissue. In these cells, Orai3 exhibits a cell-specific relevance in Ca2+ influx. In estrogen receptor-positive breast cancer cells and non-small cell lung cancer (NSCLC) cells store-operated Ca2+ entry (SOCE) is strongly dependent on Orai3 expression while in colorectal cancer and pancreatic adenocarcinoma cells Orai3 predominantly modulates SOCE. On the other hand, in prostate cancer cells Orai3 expression has been associated with the formation of Orai1/Orai3 heteromeric channels regulated by AA and reduction in SOCE, thus leading to enhanced proliferation. Orai3 overexpression is associated with supporting several cancer hallmarks, including cell cycle progression, proliferation, migration, and apoptosis resistance. This review summarizes the current knowledge concerning the functional role of Orai3 in the pathogenesis of cancer.

Endothelial cells (ECs) exhibit phenotypic and functional tissue specificities, crit-ical for studies in the vascular field and beyond. Thus, tissue-specific methods for isolation of highly purified ECs are necessary. Kidney, spleen, and testis ECs are relevant players in health and diseases such as chronic kidney disease, acute kid-ney injury, myelofibrosis, and cancer. Here, we provide tailored protocols for rapid and reproducible EC purification established for scRNA sequencing from these adult murine tissues using the combination of magnetic-and fluores-cence-activated cell sorting. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020) and Dumas et al. (2020).

Episodic future thinking (EFT) has been suggested to underlie anticipatory pleasure (AP), itself known to play a crucial role in social functioning (SF). Both AP and SF are impaired in various clinical populations, including autism spectrum disorders (ASD) and 22q11.2 deletion syndrome (22q11DS). Therefore, the relationship between EFT, AP and SF was investigated, as well as the potential role of projecting oneself in a social vs. non-social context. Seventy-seven participants [24 with 22q11DS, 20 with ASD, 33 typically developing controls (TDs)] (aged 12-25) were included. They were assessed with a future thinking task in which they were asked to recall a memory and produce a likely event. Narratives were rated based of specificity, richness and imaginability. Participants completed questionnaires assessing AP and SF. Narratives from ASD and 22q11DS participants were rated as less vivid compared to TDs. However, the characteristics of the narratives differed between ASD and 22q11DS participants in terms of specificity and level of details, as well as in reaction to social condition. Moreover, correlations were found between AP and EFT in both ASD and 22q11DS participants, and between SF and EFT in ASD participants. These results point towards impairments in EFT in both ASD and 22q11DS participants but with a specific profile in each condition. The observed associations between EFT and AP suggest that decreased autonoetic consciousness might underlie AP impairments. In ASD individuals, the association between SF and EFT highlights the need to better characterize EFT since EFT could be another mechanism contributing to social difficulties.

Changes in cytosolic free Ca2+ concentration play a central role in many fundamental cellular processes including muscle contraction, neurotransmission, cell proliferation, differentiation, gene transcription and cell death. Many of these processes are known to be regulated by store-operated calcium channels (SOCs), among which ORAI1 is the most studied in cancer cells, leaving the role of other ORAI channels yet inadequately addressed. Here we demonstrate that ORAI3 channels are expressed in both normal (HPDE) and pancreatic ductal adenocarcinoma (PDAC) cell lines, where they form functional channels, their knockdown affecting store operated calcium entry (SOCE). More specifically, ORAI3 silencing increased SOCE in PDAC cell lines, while decreasing SOCE in normal pancreatic cell line. We also show the role of ORAI3 in proliferation, cell cycle, viability, mitotic catastrophe and cell death. Finally, we demonstrate that ORAI3 silencing impairs pancreatic tumor growth and induces cell death in vivo, suggesting that ORAI3 could represent a potential therapeutic target in PDAC treatment.

Endothelial cells (ECs) harbor distinct phenotypical and functional characteristics depending on their tissue localization and contribute to brain, eye, lung, and muscle diseases such as dementia, macular degeneration, pulmonary hypertension, and sarcopenia. To study their function, isolation of pure ECs in high quantities is crucial. Here, we describe protocols for rapid and reproducible blood vessel EC purification established for scRNA sequencing from murine tissues using mechanical and enzymatic digestion followed by magnetic and fluorescence-activated cell sorting. For complete details on the use and execution of these protocol, please refer to Kalucka et al. (2020), Rohlenova et al. (2020), and Goveia et al. (2020).

Endothelial cells (ECs) from the small intestine, colon, liver, and heart have distinct phenotypes and functional adaptations that are dependent on their physiological environment. Gut ECs adapt to low oxygen, heart ECs to contractile forces, and liver ECs to low flow rates. Isolating high-purity ECs in sufficient quantities is crucial to study their functions. Here, we describe protocols combining magnetic and fluorescent activated cell sorting for rapid and reproducible EC purification from four adult murine tissues. For complete details on the use and execution of these protocols, please refer to Kalucka et al. (2020).

Mitochondria are important cell death checkpoints, and mitochondrial Ca2+ overload is considered as a potent apoptotic intrinsic pathway inducer. Here, we report that this Ca2+ apoptosis link is largely ineffective in inducing cell-death just by itself and required a concomitant inhibition of autophagy to counteract its pro-survival action. In such condition, an acute mitochondrial stress revealed by a DRP1-mediated mitochondrial dynamic remodeling is observed concomitantly with mitochondrial depolarization, release of cytochrome c, and efficient apoptosis induction. We also uncover that mitochondrial Ca2+ status modulates the function of autophagy as a sensitizer for chemotherapies. This priming mediated by mitochondrial Ca2+ overload and inhibition of autophagy sensitizes many cancer cells types to different chemotherapies with independent mechanisms of action. Collectively, our results redefine an important cell signaling pathway, uncovering new combined therapies for the treatment of diseases associated with mitochondrial Ca2+ homeostasis disorders such as cancer.

The heterogeneity of endothelial cells (ECs) across tissues remains incompletely inventoried. We constructed an atlas of >32,000 single-EC transcriptomes from 11 mouse tissues and identified 78 EC subclusters, including Aqp7+ intestinal capillaries and angiogenic ECs in healthy tissues. ECs from brain/testis, liver/spleen, small intestine/colon, and skeletal muscle/heart pairwise expressed partially overlapping marker genes. Arterial, venous, and lymphatic ECs shared more markers in more tissues than did heterogeneous capillary ECs. ECs from different vascular beds (arteries, capillaries, veins, lymphatics) exhibited transcriptome similarity across tissues, but the tissue (rather than the vessel) type contributed to the EC heterogeneity. Metabolic transcriptome analysis revealed a similar tissue-grouping phenomenon of ECs and heterogeneous metabolic gene signatures in ECs between tissues and between vascular beds within a single tissue in a tissue-type-dependent pattern. The EC atlas taxonomy enabled identification of EC subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

Since last decade real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest. Indeed, using qPCR, expression profile analyses are now possible and participate to the understanding of physiological or pathological role of channels such as calcium release-activated channels (CRAC). Initially discovered in lymphocyte T and immunity perturbations, recent studies have highlighted the role of CRAC channels in other pathologies such as cancer. Here we describe a protocol to quantify CRAC components expression, in tissue sample and cell lines, to validate knockdown strategies or identify their roles in physiological and pathological conditions (Hoth and Penner, J Physiol 465:359-386, 1993; Hoth and Penner, Nature 355:353-356, 1992; and Zweifach and Lewis, Proc Natl Acad Sci U S A 90:6295-6299, 1993).

Purpose Malignant hyperthermia (MH) is a pharmacogenetic disorder arising from uncontrolled muscle calcium release due to an abnormality in the sarcoplasmic reticulum (SR) calcium-release mechanism triggered by halogenated inhalational anesthetics. However, the molecular mechanisms involved are still incomplete. Methods We aimed to identify transient receptor potential vanilloid 1 ( TRPV1 ) variants within the entire coding sequence in patients who developed sensitivity to MH of unknown etiology. In vitro and in vivo functional studies were performed in heterologous expression system, trpv1 −/− mice, and a murine model of human MH. Results We identified TRPV1 variants in two patients and their heterologous expression in muscles of trpv1 −/− mice strongly enhanced calcium release from SR upon halogenated anesthetic stimulation, suggesting they could be responsible for the MH phenotype. We confirmed the in vivo significance by using mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation associated with MH in humans. We showed that the TRPV1 antagonist capsazepine slows the heat-induced hypermetabolic response in this model. Conclusion We propose that TRPV1 contributes to MH and could represent an actionable therapeutic target for prevention of the pathology and also be responsible for MH sensitivity when mutated.

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASN(KD)) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASN(KD) elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASN(KD) ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade.

The characterization of protein-protein interactions through methods such as co-immunoprecipitation, followed by Western blot analysis, is a crucial step in the understanding of protein functions and the biology of the cell. Since the discovery of ORAI and STIM proteins as component of store-operated channel (SOC), overexpressing systems have been used to demonstrate how ORAI and STIM can associate with physiological and pathological conditions. Here we describe a protocol allowing endogenous studies.

The role of phosphoglycerate dehydrogenase (PHGDH), a key enzyme of the serine synthesis pathway (SSP), in endothelial cells (ECs) remains poorly characterized. We report that mouse neonates with EC-specific PHGDH deficiency suffer lethal vascular defects within days of gene inactivation, due to reduced EC proliferation and survival. In addition to nucleotide synthesis impairment, PHGDH knockdown (PHGDHKD) caused oxidative stress, due not only to decreased glutathione and NADPH synthesis but also to mitochondrial dysfunction. Electron transport chain (ETC) enzyme activities were compromised upon PHGDHKD because of insufficient heme production due to cellular serine depletion, not observed in other cell types. As a result of heme depletion, elevated reactive oxygen species levels caused EC demise. Supplementation of hemin in PHGDHKD ECs restored ETC function and rescued the apoptosis and angiogenesis defects. These data argue that ECs die upon PHGDH inhibition, even without external serine deprivation, illustrating an unusual importance of serine synthesis for ECs.

Little is known about the metabolism of quiescent endothelial cells (QECs). Nonetheless, when dysfunctional, QECs contribute to multiple diseases. Previously, we demonstrated that proliferating endothelial cells (PECs) use fatty acid β-oxidation (FAO) for de novo dNTP synthesis. We report now that QECs are not hypometabolic, but upregulate FAO >3-fold higher than PECs, not to support biomass or energy production but to sustain the tricarboxylic acid cycle for redox homeostasis through NADPH regeneration. Hence, endothelial loss of FAO-controlling CPT1A in CPT1AΔEC mice promotes EC dysfunction (leukocyte infiltration, barrier disruption) by increasing endothelial oxidative stress, rendering CPT1AΔEC mice more susceptible to LPS and inflammatory bowel disease. Mechanistically, Notch1 orchestrates the use of FAO for redox balance in QECs. Supplementation of acetate (metabolized to acetyl-coenzyme A) restores endothelial quiescence and counters oxidative stress-mediated EC dysfunction in CPT1AΔEC mice, offering therapeutic opportunities. Thus, QECs use FAO for vasculoprotection against oxidative stress-prone exposure.

Glutamine synthetase, encoded by the gene GLUL , is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.