TMEM16F exacerbates tau pathology and mediates phosphatidylserine exposure in phospho-tau-burdened neurons.

TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane. While the functions of TMEM16F have been extensively characterized in multiple cell types, the role of TMEM16F in the central nervous system remains largely unknown. Here, we sought to study how TMEM16F in the brain may be involved in neurodegeneration. Using a mouse model that expresses the pathological P301S human tau (PS19 mouse), we found reduced tauopathy and microgliosis in 6- to 7-mo-old PS19 mice lacking TMEM16F. Furthermore, this reduction of pathology can be recapitulated in the PS19 mice with TMEM16F removed from neurons, while removal of TMEM16F from microglia of PS19 mice did not significantly impact tauopathy at this time point. Moreover, TMEM16F mediated aberrant phosphatidylserine exposure in neurons with phospho-tau burden. These studies raise the prospect of targeting TMEM16F in neurons as a potential treatment of neurodegeneration.

Study on the Mechanism of the Adrenaline-Evoked Procoagulant Response in Human Platelets.

Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the mechanism is only fragmentarily established. Using a panel of platelet receptors' antagonists and modulators of signaling pathways, we evaluated the importance of these in adrenaline-evoked PS exposure by flow cytometry. Calcium and sodium ion influx into platelet cytosol, after adrenaline treatment, was examined by fluorimetric measurements. We found a strong reduction in PS exposure after blocking of sodium and calcium ion influx via Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX), respectively. ADP receptor antagonists produced a moderate inhibitory effect. Substantial limitation of PS exposure was observed in the presence of GPIIb/IIIa antagonist, phosphoinositide-3 kinase (PI3-K) inhibitors, or prostaglandin E1, a cyclic adenosine monophosphate (cAMP)-elevating agent. We demonstrated that adrenaline may develop a procoagulant response in human platelets with the substantial role of ion exchangers (NHE and NCX), secreted ADP, GPIIb/IIIa-dependent outside-in signaling, and PI3-K. Inhibition of the above mechanisms and increasing cytosolic cAMP seem to be the most efficient procedures to control adrenaline-evoked PS exposure in human platelets.

Silica nanoparticles trigger phosphatidylserine exposure in red blood cells and induce thrombosis risk.

Silica nanoparticles (SiNPs) have attracted increasing attention for their health effects due to the increased risk of exposure to human bodies via diverse routes. Considering that SiNPs enter the circulatory system and inevitably encounter red blood cells (RBCs), it is necessary to investigate their risk of causing erythrocytotoxicity. In this study, three sizes of SiNPs (SiNP-60, SiNP-120, and SiNP-200) were tested for their effects on mouse RBCs. The results showed that SiNPs could induce hemolysis, morphological changes, and phosphatidylserine (PS) exposure in RBCs in a particulate size-related manner. Further investigations on the underlying mechanism indicated that SiNP-60 exposure increased intracellular reactive oxidative species (ROS) generation and subsequently caused the phosphorylation of p38 and ERK1/2 in RBCs. The addition of antioxidants or inhibitors of mitogen-activated protein kinase (MAPK) signaling significantly attenuated PS exposure in RBCs and ameliorated SiNP-induced erythrocytotoxicity. Moreover, ex vivo assays using platelet-rich plasma (PRP) showed that SiNP-60-induced PS exposure in RBCs could trigger thrombin-dependent platelet activation. The contrary evidence from the assays of PS blockage and thrombin inhibition further confirmed that SiNP-60-induced platelet activation was dependent on PS externalization in RBCs, concomitantly with thrombin formation. These findings revealed the procoagulant and prothrombotic effects of SiNPs through the regulation of PS externalization in RBCs, and may be of great help in bridging the knowledge gap on the potential cardiovascular hazards of particulate silica from both artificial and naturally occurring origins.

Myricetin-induced suicidal erythrocyte death.

BACKGROUND: Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. METHODS AND RESULTS: Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24 h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. CONCLUSION: Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

Electric Fields Regulate In Vitro Surface Phosphatidylserine Exposure of Cancer Cells via a Calcium-Dependent Pathway.

Cancer is the second leading cause of death worldwide after heart disease. The current treatment options to fight cancer are limited, and there is a critical need for better treatment strategies. During the last several decades, several electric field (EF)-based approaches for anti-cancer therapies have been introduced, such as electroporation and tumor-treating fields; still, they are far from optimal due to their invasive nature, limited efficacy and significant side effects. In this study, we developed a non-contact EF stimulation system to investigate the in vitro effects of a novel EF modality on cancer biomarkers in normal (human astrocytes, human pancreatic ductal epithelial -HDPE-cells) and cancer cell lines (glioblastoma U87-GBM, human pancreatic cancer cfPac-1, and MiaPaCa-2). Our results demonstrate that this EF modality can successfully modulate an important cancer cell biomarker-cell surface phosphatidylserine (PS). Our results further suggest that moderate, but not low, amplitude EF induces p38 mitogen-activated protein kinase (MAPK), actin polymerization, and cell cycle arrest in cancer cell lines. Based on our results, we propose a mechanism for EF-mediated PS exposure in cancer cells, where the magnitude of induced EF on the cell surface can differentially regulate intracellular calcium (Ca2+) levels, thereby modulating surface PS exposure.

Hyperinflammation, apoptosis, and organ damage.

The cytokine storm (CS) in hyperinflammation is characterized by high levels of cytokines, extreme activation of innate as well as adaptive immune cells and initiation of apoptosis. High levels of apoptotic cells overwhelm the proper recognition and removal system of these cells. Phosphatidylserine on the apoptotic cell surface, which normally provides a recognition signal for removal, becomes a target for hemostatic proteins and secretory phospholipase A2. The dysregulation of these normal pathways in hemostasis and the inflammasome result in a prothrombotic state, cellular death, and end-organ damage. In this review, we provide the argument that this imbalance in recognition and removal is a common denominator regardless of the inflammatory trigger. The complex reaction of the immune defense system in hyperinflammation leads to self-inflicted damage. This common endpoint may provide additional options to monitor the progression of the inflammatory syndrome, predict severity, and may add to possible treatment strategies.

Eryptosis: Programmed Death of Nucleus-Free, Iron-Filled Blood Cells.

Human erythrocytes are organelle-free cells packaged with iron-containing hemoglobin, specializing in the transport of oxygen. With a total number of approximately 25 trillion cells per individual, the erythrocyte is the most abundant cell type not only in blood but in the whole organism. Despite their low complexity and their inability to transcriptionally upregulate antioxidant defense mechanisms, they display a relatively long life time, of 120 days. This ensures the maintenance of tissue homeostasis where the clearance of old or damaged erythrocytes is kept in balance with erythropoiesis. Whereas the regulatory mechanisms of erythropoiesis have been elucidated over decades of intensive research, the understanding of the mechanisms of erythrocyte clearance still requires some refinement. Here, we present the main pathways leading to eryptosis, the programmed death of erythrocytes, with special emphasis on Ca2+ influx, the generation of ceramide, oxidative stress, kinase activation, and iron metabolism. We also compare stress-induced erythrocyte death with erythrocyte ageing and clearance, and discuss the similarities between eryptosis and ferroptosis, the iron-dependent regulated death of nucleated blood cells. Finally, we focus on the pathologic consequences of deranged eryptosis, and discuss eryptosis in the context of different infectious diseases, e.g., viral or parasitic infections, and hematologic disorders.

The ratio of ATP11C/PLSCR1 mRNA transcripts has clinical significance in sickle cell anemia.

One of the physiologic mechanisms responsible to maintain asymmetric phospholipid distribution (in particular phosphatidylserine, PS) in human erythrocyte membranes is orchestrated by the balance between enzymes responsible for active transport of PS from the outer to the inner leaflet (ATP11C) and those whose counteracts these activities (PLSCR1). Using quantitative real-time polymerase chain reaction and standard flow cytometry procedures, we hypothesized that the aberrant expression of either or both ATP11C and PLSCR1 transcripts may disrupt the PS internalization/externalization process and become clinically relevant for patients with sickle cell anemia (SCA). Overall, neither ATP11C/PLSCR1 ratio or ATP11C and PLSCR1 (if analyzed separately) had impact on risk to present acute or chronic organ damage in 178 patients with SCA. By collecting a new set of samples from SCA patients during a vaso-occlusive crisis (VOC, crisis state, 13 patients) and comparing with new samples of patients in steady state (15 patients), we noticed that patients in steady state exhibited mean values of ATP11C/PLSCR1 ratio significantly higher (mean value: 18.2, range, 0.3-53) than those who were in crisis (mean value: 3.7, range, 0.5-9) (P = 0.013). Most importantly, there was a strong inverse correlation between PS exposure and ATP11C/PLSCR1 ratio in sickle erythrocytes (Pearson correlation coefficient, r: - 0.78). Based on these findings, it is conceivable that the ATP11C/PLSCR1 ratio may switch from high to low during a VOC, although the underlying reasons require further investigations.

Enhanced Eryptosis in Glucose-6-Phosphate Dehydrogenase Deficiency.

BACKGROUND/AIMS: Defects in the Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme enhance cellular oxidative damage, thus impairing erythrocytes and radically shortening their lifespan. We aimed to study programmed erythrocyte cell death in G6PD-deficient patients, describe the molecular genetics basis of G6PD and investigate phenotype-genotype correlations. METHODS: We explored eryptosis using the annexin V-binding assay, taken as an indicator of PS exposure at the erythrocyte surface. We assessed reactive oxygen species (ROS) production, intracellular calcium concentrations and ceramide formation at the cell surface. Prior to and following treatments, cells were analyzed by flow cytometry. Finally, we explored G6PD gene mutations through PCR-Sanger sequencing. RESULTS: Before stimulation, PS-exposing erythrocytes were significantly higher in G6PD-deficient patients than in healthy volunteers. This was paralleled by a significant increase in reactive oxygen species production, suggesting that oxidative stress is the main trigger of PS exposure in G6PD-deficient erythrocytes. Five previously described mutations were detected in our patients. Two genotypes correlated with a significantly higher percentage of PS-exposing cells. CONCLUSION: Our study uncovers a novel effect detected in G6PD-deficient erythrocytes which is cell membrane scrambling with PS translocation to the erythrocyte surface. Our findings shed a light on the mechanisms of premature erythrocyte clearance in G6PD deficiency.

Adrenaline May Contribute to Prothrombotic Condition via Augmentation of Platelet Procoagulant Response, Enhancement of Fibrin Formation, and Attenuation of Fibrinolysis.

Background: Adrenaline is believed to play a role in thrombosis and hemostasis. The complex effect of its clinically relevant concentrations on thrombus formation, coagulation and fibrinolysis in human blood has never been specifically studied. Methods: Confocal microscopy was used to study thrombus formation under flow, exposure of phosphatidylserine (PS) in adhered platelets, to evaluate clots density, and to measure kinetics of fibrin formation and external fibrinolysis under flow. Flow cytometry was utilized to assess PS exposure in non-adhered platelets. Kinetics of clot formation and internal fibrinolysis was evaluated by thromboelastometry. Platelet aggregation was measured by optical aggremometry. Kinetics of clot retraction was assessed by using digital camera. Results: We found that adrenaline (1-10 nM) is able to enhance platelet activation evoked by subthreshold collagen (150 ng/ml), resulting in augmentation of platelet aggregation, thrombus formation under arterial flow conditions, platelet PS exposure, and formation of platelet-fibrin clots. The development of platelet procoagulant response evoked by adrenaline + low collagen was associated with the formation of denser platelet-fibrin clots and the decrease in rate of fibrinolysis despite whether lysis was initiated inside (internal fibrinolysis) or outside the clot (external fibrinolysis). The above phenomena were abolished by the α2-adrenergic receptor antagonist, rauwolscine. Adrenaline-collagen synergism, expressed as PS exposure, was significantly reduced by cyclooxygenase inhibitor (acetylsalicic acid), GPIIb/IIIa receptor blocker (tirofiban), and P2Y12 receptor antagonist (PSB 0739). Conclusion: Clinically relevant concentrations of adrenaline may significantly augment responses of human platelets in the presence of subthreshold concentrations of collagen, which should be considered during therapies involving adrenaline infusion. Routinely used antiplatelet drugs may reduce the prothrombotic state evoked by adrenaline-collagen synergism.

Eryptosis and Malaria: New Experimental Guidelines and Re-Evaluation of the Antimalarial Potential of Eryptosis Inducers.

Erythrocytes possess an unusual programmed cell death mechanism termed eryptosis, and several compounds have been previously claimed to induce eryptosis in vitro. Malaria parasites (genus Plasmodium) reside in erythrocytes during the pathogenic part of their life cycle, and the potential of several eryptosis inducers to act as antimalarials has been tested in recent years. However, the eryptosis-inducing capacity of these compounds varies significantly between eryptosis-focused studies and malaria investigations. Here, we investigated the reasons for these discrepancies, we developed a protocol to investigate eryptosis in malaria cultures and we re-evaluated the potential of eryptosis inducers as antimalarials. First, we showed that eryptosis read-out in vitro is dependent on culture conditions. Indeed, conditions that have consistently been used to study eryptosis do not support P. falciparum growth and prime erythrocytes for eryptosis. Next, we defined culture conditions that allow the detection of eryptosis while supporting P. falciparum survival. Finally, we selected six eryptosis-inducers based on their clinical use, molecular target and antimalarial activities, and re-evaluated their eryptosis inducing capacities and their potential as antimalarials. We demonstrate that none of these compounds affect the viability of naïve or P. falciparum-infected erythrocytes in vitro. Nevertheless, three of these compounds impair parasite development, although through a mechanism unrelated to eryptosis and yet to be elucidated. We conclude that careful consideration of experimental set up is key for the accurate assessment of the eryptosis-inducing potential of compounds and their evaluation as potential antimalarials.

Of membranes and malaria: phospholipid asymmetry in Plasmodium falciparum-infected red blood cells.

Malaria is a vector-borne parasitic disease with a vast impact on human history, and according to the World Health Organisation, Plasmodium parasites still infect over 200 million people per year. Plasmodium falciparum, the deadliest parasite species, has a remarkable ability to undermine the host immune system and cause life-threatening disease during blood infection. The parasite's host cells, red blood cells (RBCs), generally maintain an asymmetric distribution of phospholipids in the two leaflets of the plasma membrane bilayer. Alterations to this asymmetry, particularly the exposure of phosphatidylserine (PS) in the outer leaflet, can be recognised by phagocytes. Because of the importance of innate immune defence numerous studies have investigated PS exposure in RBCs infected with P. falciparum, but have reached different conclusions. Here we review recent advancements in our understanding of the molecular mechanisms which regulate asymmetry in RBCs, and whether infection with the P. falciparum parasite results in changes to PS exposure. On the balance of evidence, it is likely that membrane asymmetry is disrupted in parasitised RBCs, though some methodological issues need addressing. We discuss the potential causes and consequences of altered asymmetry in parasitised RBCs, particularly for in vivo interactions with the immune system, and the role of host-parasite co-evolution. We also examine the potential asymmetric state of parasite membranes and summarise current knowledge on the parasite proteins, which could regulate asymmetry in these membranes. Finally, we highlight unresolved questions at this time and the need for interdisciplinary approaches to uncover the machinery which enables P. falciparum parasites to hide in mature erythrocytes.

Plasma membrane lipid scrambling causing phosphatidylserine exposure negatively regulates NK cell activation.

One of the hallmarks of live cells is the asymmetric distribution of lipids across their plasma membrane. Changes in this asymmetry due to lipid "scrambling" result in phosphatidylserine exposure at the cell surface that is detected by annexin V staining. This alteration is observed during cell death processes such as apoptosis, and during physiological responses such as platelet degranulation and membrane repair. Previous studies have shown that activation of NK cells is accompanied by exposure of phosphatidylserine at the cell surface. While this response was thought to be indicative of ongoing NK cell death, it may also  reflect the regulation of NK cell activation in the absence of cell death. Herein, we found that NK cell activation was accompanied by rapid phosphatidylserine exposure to an extent proportional to the degree of NK cell activation. Through enforced expression of a lipid scramblase, we provided evidence that activation-induced lipid scrambling in NK cells is reversible and does not lead to cell death. In contrast, lipid scrambling attenuates NK cell activation. This response was accompanied by reduced cell surface expression of activating receptors such as 2B4, and by loss of binding of Src family protein tyrosine kinases Fyn and Lck to the inner leaflet of the plasma membrane. Hence, lipid scrambling during NK cell activation is, at least in part, a physiological response that reduces the NK cell activation level. This effect is due to the ability of lipid scrambling to alter the distribution of membrane-associated receptors and kinases required for NK cell activation.

Type 1 interferon aggravates lipopolysaccharide-induced sepsis through upregulating Caspase-11 and Gasdermin D.

This study aimed to investigate the mechanism of type I interferon (IFN) in aggravating sepsis in bacterial infection, focusing on the roles of Caspase-11 (Casp11) and Gasdermin D (Gsdmd) in this process. Type I interferons, including IFNα and IFNß, were used to treat peritoneal macrophage harvested from wild-type or IFNα/ßR1 knockout (KO) mice, of which the levels of Casp11 and Gsdmd were monitored using real-time polymerase chain reaction (RT-PCR) and Western blot, the exposure to phosphatidylserine was monitored by flow cytometry, and tissue factor (TF) activation was assessed by RT-PCR and TF chromogenic assay. Endotoxemia in wild-type mice led to upregulation of Casp11 and Gsdmd in myeloid cells, which in contrast was attenuated in IFNα/ßR1 KO mice. IFNα or IFNß treatment led to dose-dependent upregulation of Casp11 and Gsdmd in peritoneal macrophages harvested from wild-type mice, but induced negligible changes in IFNα/ßR1 KO mice. Type I IFN promoted phosphatidylserine exposure in peritoneal macrophage from wild-type mice but not IFNα/ßR1 KO mice. Type I IFN induced insignificant changes of TF expression levels in both wild-type mice and IFNα/ßR1 KO mice, but the TF activity was markedly increased in wild-type mice after type I IFN treatment. Our data suggested that the upregulation of Casp11 and Gsdmd in myeloid cells and macrophages induced by endotoxemia was reliant on the expression of IFNα/ßR1. IFNα or IFNß treatment efficiently upregulated Casp11 and Gsdmd, phosphatidylserine exposure, and TF activity of macrophages. Therefore, type I IFN could aggravate sepsis through upregulating Casp11 and Gsdmd.

Cargo-laden erythrocyte ghosts target liver mediated by macrophages.

Liver-targeted cargo delivery possesses great potential for the treatment of liver disease. It is urgent to find an efficient and biocompatible liver targeted delivery system. This study focused on the liver targeting properties of erythrocyte ghosts and its possible mechanism. Herein, we optimized conditions to fabricate human and mouse erythrocyte ghosts with sufficient room capable of incorporating various model substances. Erythrocyte ghosts are biocompatible cargo carriers because it is derived from autologous red blood cells (RBCs), and the cell size, zeta potential, and biconcave-disk shape of the ghosts were consistent with those of RBCs. An in vivo imaging system and positron emission tomography/computed tomography imaging showed that the ghosts were captured mainly in the liver by intravenous injection of fluorescence or 18F-fluorodeoxyglucose (FDG)-labelled ghosts into mice. In contrast, the main concentration of naked octreotide was trapped in the lungs while naked 18F-FDG was trapped in the heart. However, the concentration of cargo-loaded ghosts decreased significantly in the liver in macrophage-depleted mice. Accordingly, in vitro experiments showed that higher phosphatidylserine exposure was observed in the ghosts (38.9 %) compared to normal erythrocytes (0.69 %), and the phagocytic activity of the macrophage RAW 264.7. on the ghosts was significantly higher than that of normal erythrocytes (p < 0.001). Together they indicate that erythrocyte ghosts show liver targeting properties, and possibly owing to macrophage phagocytosis. This promising and effective therapeutic delivery system may provide therapeutic benefits for liver disease.

Nanoscale adhesion profiling and membrane characterisation in sickle cell disease using hybrid atomic force microscopy-IR spectroscopy.

Sickle cell disease (SCD) presents a significant global health problem. At present there is no effective treatment, with most being supportive for its associated complications such as the vaso-occlusive crises that result from increased cell adhesion. Hypoxic sickle cells have previously shown greater phosphatidylserine (PS) exposure and oxidative damage, as well as being notably "stickier" suggesting that increased cell cohesion and adhesion to the blood vessel endothelium is a possible mechanism for vaso-occlusion. The present work uses the hybrid technique of atomic force microscopy nano-infrared spectroscopy (AFM-IR) to probe changes to the coefficient of friction and C-O IR intensity in SCD on a nanoscale for dried red blood cells (RBCs) fixed under conditions of hypoxia and correlates these observations with adhesive interactions at the membrane. Using functionalised AFM tips, it has been possible to probe adhesive interactions between hydrophilic and hydrophobic moieties exposed at the surface of the dried RBCs fixed under different oxygenation states and for different cell genotypes. The results are consistent with greater PS-exposure and oxidative damage in hypoxic sickle cells, as previously proposed, and also show strong correlation between localised oxidative damage and increased adhesion. A mechanistic explanation involving significant lipid tail disruption as a result of oxidative action, in combination with differing concentrations of externalised PS lipids, is proposed to explain the observed adhesion behaviour of each type of cell.

HAuCl4, Putative General Aquaporins Blocker, Reduces Platelet Spreading, Filopodia Formation, Procoagulant Response, and Thrombus Formation Under Flow.

Background: Recent studies indicate that aquaporin (AQP) water channels have a regulatory function in human platelet secretion and in procoagulant response of murine platelets. However, the engagement of AQPs in morphological changes, procoagulant response, and thrombus formation in human blood has never been investigated. Methods: Confocal microscopy was used to study platelet spreading, filopodia formation, ballooning, and thrombus formation under flow. Flow cytometry was utilized to assess platelet phosphatidylserine (PS) exposure and microparticles shedding. Kinetics of clot formation in vitro was evaluated by thromboelastometry. Mouse model of ferric chloride (III) (FeCl3)-induced thrombosis was used to investigate thrombus formation in vivo. Results: We found that chloroauric(III) acid (HAuCl4), a classical AQP inhibitor (10-100 µM), reduced spreading of human platelets on collagen-coated surfaces and inhibited filopodia formation in a fluid phase. Under flow conditions, HAuCl4 (100 µM) attenuated thrombi growth on collagen, platelet secretion, and PS exposure. Thrombus formation was restored by the addition of exogenous adenosine diphosphate (ADP). Collagen-evoked platelet procoagulant response (evaluated as PS exposure, shedding of microparticles, platelet-dependent thrombin generation, and membrane ballooning) was distinctly reduced by HAuCl4 (25-200 µM), as well as the dynamics of clot formation. In mouse model of thrombosis, reduction of surface of PS-positive cells within thrombus was observed in the presence of HAuCl4 (1-10 mg/kg). Conclusion: These results suggest that in human platelets AQPs are crucial for agonist-evoked morphological changes, thrombus formation under flow, and in development of procoagulant response. Antithrombotic effect in vivo suggests that nontoxic inhibitors of AQPs may be considered as potential candidates for a novel class of antiplatelet drugs.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation.

Plasma membrane damage and cell death during processes such as necroptosis and apoptosis result from cues originating intracellularly. However, death caused by pore-forming agents, like bacterial toxins or complement, is due to direct external injury to the plasma membrane. To prevent death, the plasma membrane has an intrinsic repair ability. Here, we found that repair triggered by pore-forming agents involved TMEM16F, a calcium-activated lipid scramblase also mutated in Scott's syndrome. Upon pore formation and the subsequent influx of intracellular calcium, TMEM16F induced rapid "lipid scrambling" in the plasma membrane. This response was accompanied by membrane blebbing, extracellular vesicle release, preserved membrane integrity, and increased cell viability. TMEM16F-deficient mice exhibited compromised control of infection by Listeria monocytogenes associated with a greater sensitivity of neutrophils to the pore-forming Listeria toxin listeriolysin O (LLO). Thus, the lipid scramblase TMEM16F is critical for plasma membrane repair after injury by pore-forming agents.

In vitro bioactivity approach of unripe genipap (Genipa americana L., Rubiaceae) fruit extract and its solid lipid microparticle.

Growing awareness in favor of innovative and healthier alternatives is creating a noticeable shift from synthetic colorants to natural additives. And, such a swing in the consumer market is growing slowly but noticeably. In this context, genipap (Genipa americana L.) fruit represents an emerging source of blue colorants in Latin America with extensive application possibilities. This is despite the fact that there are few studies concerning its toxicity predictive factors. In this early-stage study we propose to investigate safety issues around genipap extract (IBBP); we also attempt to identify fingerprint profiling of both IBBP extract and solid lipid microparticles containing IBBP extract (SLM-IBBP) using in vitro assays. The main compounds identified were genipin, and genipin 1-ß-gentiobioside. Results indicated that IBBP extract, at 25 µg/mL, was able to promote DNA damage in CHO-K1 cells, suggesting a genotoxic effect. On the other hand, the SLM-IBBP inhibited almost all cancer cell lines with GI50 ranging from 0.25 µg/mL to 43.5 µg/mL. Also, IBBP-SLM seems to exert a desirable apoptosis induction (at 25 µg/mL dosage). The next steps for our work, therefore, will focus on other nanoparticle formulation approaches, in particular with the use of natural Brazilian starch. An evaluation of the metabolism and distribution of microparticles, and their safety for food and pharmaceutical purposes, are also required.