A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase.

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

Phosphatidylserine-positive extracellular vesicles boost effector CD8+ T cell responses during viral infection.

CD8+ T cells are crucial for the clearance of viral infections. During the acute phase, proinflammatory conditions increase the amount of circulating phosphatidylserine+ (PS) extracellular vesicles (EVs). These EVs interact especially with CD8+ T cells; however, it remains unclear whether they can actively modulate CD8+ T cell responses. In this study, we have developed a method to analyze cell-bound PS+ EVs and their target cells in vivo. We show that EV+ cell abundance increases during viral infection and that EVs preferentially bind to activated, but not naive, CD8+ T cells. Superresolution imaging revealed that PS+ EVs attach to clusters of CD8 molecules on the T cell surface. Furthermore, EV-binding induces antigen (Ag)-specific TCR signaling and increased nuclear translocation of the transcription factor Nuclear factor of activated T-cells (NFATc1) in vivo. EV-decorated but not EV-free CD8+ T cells are enriched for gene signatures associated with T-cell receptor signaling, early effector differentiation, and proliferation. Our data thus demonstrate that PS+ EVs provide Ag-specific adjuvant effects to activated CD8+ T cells in vivo.

Phosphatidylserine clustering by the Ebola virus matrix protein is a critical step in viral budding.

Phosphatidylserine (PS) is a critical lipid factor in the assembly and spread of numerous lipid-enveloped viruses. Here, we describe the ability of the Ebola virus (EBOV) matrix protein eVP40 to induce clustering of PS and promote viral budding in vitro, as well as the ability of an FDA-approved drug, fendiline, to reduce PS clustering and subsequent virus budding and entry. To gain mechanistic insight into fendiline inhibition of EBOV replication, multiple in vitro assays were run including imaging, viral budding and viral entry assays. Fendiline lowers PS content in mammalian cells and PS in the plasma membrane, where the ability of VP40 to form new virus particles is greatly lower. Further, particles that form from fendiline-treated cells have altered particle morphology and cannot significantly infect/enter cells. These complementary studies reveal the mechanism by which EBOV matrix protein clusters PS to enhance viral assembly, budding, and spread from the host cell while also laying the groundwork for fundamental drug targeting strategies.

The Anticancer Drug Bleomycin Shows Potent Antifungal Activity by Altering Phospholipid Biosynthesis.

Invasive fungal infections are difficult to treat with limited drug options, mainly because fungi are eukaryotes and share many cellular mechanisms with the human host. Most current antifungal drugs are either fungistatic or highly toxic. Therefore, there is a critical need to identify important fungal specific drug targets for novel antifungal development. Numerous studies have shown the fungal phosphatidylserine (PS) biosynthetic pathway to be a potential target. It is synthesized from CDP-diacylglycerol and serine, and the fungal PS synthesis route is different from that in mammalian cells, in which preexisting phospholipids are utilized to produce PS in a base-exchange reaction. In this study, we utilized a Saccharomyces cerevisiae heterologous expression system to screen for inhibitors of Cryptococcus PS synthase Cho1, a fungi-specific enzyme essential for cell viability. We identified an anticancer compound, bleomycin, as a positive candidate that showed a phospholipid-dependent antifungal effect. Its inhibition on fungal growth can be restored by ethanolamine supplementation. Further exploration of the mechanism of action showed that bleomycin treatment damaged the mitochondrial membrane in yeast cells, leading to increased generation of reactive oxygen species (ROS), whereas supplementation with ethanolamine helped to rescue bleomycin-induced damage. Our results indicate that bleomycin does not specifically inhibit the PS synthase enzyme; however, it may affect phospholipid biosynthesis through disruption of mitochondrial function, namely, the synthesis of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), which helps cells maintain membrane composition and functionality. IMPORTANCE Invasive fungal pathogens cause significant morbidity and mortality, with over 1.5 million deaths annually. Because fungi are eukaryotes that share much of their cellular machinery with the host, our armamentarium of antifungal drugs is highly limited, with only three classes of antifungal drugs available. Drug toxicity and emerging resistance have limited their use. Hence, targeting fungi-specific enzymes that are important for fungal survival, growth, or virulence poses a strategy for novel antifungal development. In this study, we developed a heterologous expression system to screen for chemical compounds with activity against Cryptococcus phosphatidylserine synthase, Cho1, a fungi-specific enzyme that is essential for viability in C. neoformans. We confirmed the feasibility of this screen method and identified a previously unexplored role of the anticancer compound bleomycin in disrupting mitochondrial function and inhibiting phospholipid synthesis.

Anti-Eryptotic Activity of Food-Derived Phytochemicals and Natural Compounds.

Human red blood cells (RBCs), senescent or damaged due to particular stress, can be removed by programmed suicidal death, a process called eryptosis. There are various molecular mechanisms underlying eryptosis. The most frequent is the increase in the cytoplasmic concentration of Ca2+ ions, later exposure of erythrocytes to oxidative stress, hyperosmotic shock, ceramide formation, stimulation of caspases, and energy depletion. Phosphatidylserine (PS) exposed by eryptotic RBCs due to interaction with endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor, causes the RBCs to adhere to vascular wall with consequent damage to the microcirculation. Eryptosis can be triggered by various xenobiotics and endogenous molecules, such as high cholesterol levels. The possible diseases associated with eryptosis are various, including anemia, chronic kidney disease, liver failure, diabetes, hypertension, heart failure, thrombosis, obesity, metabolic syndrome, arthritis, and lupus. This review addresses and collates the existing ex vivo and animal studies on the inhibition of eryptosis by food-derived phytochemicals and natural compounds including phenolic compounds (PC), alkaloids, and other substances that could be a therapeutic and/or co-adjuvant option in eryptotic-driven disorders, especially if they are introduced through the diet.

Transfer of Cellular Content from the Allogeneic Cell-Based Cancer Vaccine DCP-001 to Host Dendritic Cells Hinges on Phosphatidylserine and Is Enhanced by CD47 Blockade.

DCP-001 is a cell-based cancer vaccine generated by differentiation and maturation of cells from the human DCOne myeloid leukemic cell line. This results in a vaccine comprising a broad array of endogenous tumor antigens combined with a mature dendritic cell (mDC) costimulatory profile, functioning as a local inflammatory adjuvant when injected into an allogeneic recipient. Intradermal DCP-001 vaccination has been shown to be safe and feasible as a post-remission therapy in acute myeloid leukemia. In the current study, the mode of action of DCP-001 was further characterized by static and dynamic analysis of the interaction between labelled DCP-001 and host antigen-presenting cells (APCs). Direct cell-cell interactions and uptake of DCP-001 cellular content by APCs were shown to depend on DCP-001 cell surface expression of calreticulin and phosphatidylserine, while blockade of CD47 enhanced the process. Injection of DCP-001 in an ex vivo human skin model led to its uptake by activated skin-emigrating DCs. These data suggest that, following intradermal DCP-001 vaccination, local and recruited host APCs capture tumor-associated antigens from the vaccine, become activated and migrate to the draining lymph nodes to subsequently (re)activate tumor-reactive T-cells. The improved uptake of DCP-001 by blocking CD47 rationalizes the possible combination of DCP-001 vaccination with CD47 blocking therapies.

Solidagenone acts on promastigotes of L. amazonensis by inducing apoptosis-like processes on intracellular amastigotes by IL-12p70/ROS/NO pathway activation.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the Leishmania genus. Currently, the treatment has limited effectiveness and high toxicity, is expensive, requires long-term treatment, induces significant side effects, and promotes drug resistance. Thus, new therapeutic strategies must be developed to find alternative compounds with high efficiency and low cost. Solidagenone (SOL), one of the main constituents of Solidago chilensis, has shown gastroprotective, anti-inflammatory and immunomodulatory effects. PURPOSE: This study assessed the in vitro effect of SOL on promastigotes and Leishmania amazonensis-infected macrophages, as well its microbicide and immunomodulatory mechanisms. METHODS: SOL was isolated from the roots of S. chilensis, 98% purity, and identified by chromatographic methods, and the effect of SOL on leishmanicidal activity against promastigotes in vitro, SOL-induced cytotoxicity in THP-1, J774 cells, sheep erythrocytes, and L. amazonensis-infected J774 macrophages, and the mechanisms of death involved in this action were evaluated. RESULTS: In silico predictions showed good drug-likeness potential for SOL with high oral bioavailability and intestinal absorption. SOL treatment (10-160 µM) inhibited promastigote proliferation 24, 48, and 72 h after treatment. After 24 h of treatment, SOL at the IC50 (34.5 µM) and 2 × the IC50 (69 µM) induced several morphological and ultrastructural changes in promastigotes, altered the cell cycle and cellular volume, increased phosphatidylserine exposure on the cell surface, induced the loss of plasma membrane integrity, increased the reactive oxygen species (ROS) level, induced loss of mitochondrial integrity (characterized by an apoptosis-like process), and increased the number of lipid droplets and autophagic vacuoles. Additionally, SOL induced low cytotoxicity in J774 murine macrophages (CC50 of 1587 µM), THP-1 human monocytes (CC50 of 1321 µM), and sheep erythrocytes. SOL treatment reduced the percentage of L. amazonensis-infected macrophages and the number of amastigotes per macrophage (IC50 9.5 µM), reduced TNF-α production and increased IL-12p70, ROS and nitric oxide (NO) levels. CONCLUSION: SOL showed in vitro leishmanicidal effects against the promastigotes by apoptosis-like mechanism and amastigotes by reducing TNF-α and increasing IL-12p70, ROS, and NO levels, suggesting their potential as a candidate for use in further studies on the design of antileishmanial drugs.

Anticancer potential of rhizome extract and a labdane diterpenoid from Curcuma mutabilis plant endemic to Western Ghats of India.

Zingiberaceae plants are well known for their use in ethnomedicine. Curcuma mutabilis Skornick., M. Sabu & Prasanthk., is an endemic Zingiberaceae species from Western Ghats of Kerala, India. Here, we report for the first time, the anticancer potential of petroleum ether extract from C. mutabilis rhizome (CMRP) and a novel labdane diterpenoid, (E)-14, 15-epoxylabda-8(17), 12-dien-16-al (Cm epoxide) isolated from it. CMRP was found to be a mixture of potent bioactive compounds including Cm epoxide. Both the extract and the compound displayed superior antiproliferative activity against several human cancer cell lines, without any display of cytotoxicity towards normal human cells such as peripheral blood derived lymphocytes and erythrocytes. CMRP treatment resulted in phosphatidylserine externalization, increase in the levels of intracellular ROS, Ca2+, loss of mitochondrial membrane potential as well as fragmentation of genomic DNA. Analyses of transcript profiling and immunostained western blots of extract-treated cancer cells confirmed induction of apoptosis by both intrinsic and extrinsic pathways. The purified compound, Cm epoxide, was also found to induce apoptosis in many human cancer cell types tested. Both CMRP and the Cm epoxide were found to be pharmacologically safe in terms of acute toxicity assessment using Swiss albino mice model. Further, molecular docking interactions of Cm epoxide with selected proteins involved in cell survival and death were also indicative of its druggability. Overall, our findings reveal that the endemic C. mutabilis rhizome extract and the compound Cm epoxide isolated from it are potential candidates for development of future cancer chemotherapeutics.

Effects of Dietary Supplementation with EPA-enriched Phosphatidylcholine and Phosphatidylethanolamine on Glycerophospholipid Profile in Cerebral Cortex of SAMP8 Mice fed with High-fat Diet.

The destruction of lipid homeostasis is associated with nervous system diseases such as Alzheimer's disease (AD). It has been reported that dietary EPA-enriched phosphatidylcholine (EPA-PC) and phosphatidylethanolamine (EPA-PE) could improve brain function. However, it was unclear that whether EPA-PC and EPA-PE intervention could change the lipid composition of cerebral cortex in AD mice. All the senescence-accelerated mouse-prone 8 (SAMP8) mice were fed with a high-fat diet for 8 weeks. After another 8 weeks of intervention with EPA-PC and EPA-PE (1%, w/w), the cerebral cortex lipid levels were determined by lipidomics. Results demonstrated that dietary supplementation with EPA-PE and EPA PC for 8 weeks significantly increased the amount of choline plasmalogen (pPC) and Lyso phosphatidylethanolamine (LPE) in the cerebral cortex of SAMP8 mice fed with high fat diet. Meanwhile, administration with EPA-PE and EPA-PC could significantly decrease the level of docosapentaenoic acid (DPA)-containing phosphatidylserine (PS) as well as increase the levels of arachidonic acid (AA)-containing phosphatidylethanolamine and PS in cerebral cortex. EPA-PE and EPA-PC could restore the lipid homeostasis of dementia mice to a certain degree, which might provide a potential novel therapy strategy and direction of dietary intervention in patients with cognitive impairment.

Muscle metabolic remodelling patterns in Duchenne muscular dystrophy revealed by ultra-high-resolution mass spectrometry imaging.

Duchenne muscular dystrophy (DMD) is a common and severe X-linked myopathy, characterized by muscle degeneration due to altered or absent dystrophin. DMD has no effective cure, and the underlying molecular mechanisms remain incompletely understood. The aim of this study is to investigate the metabolic changes in DMD using mass spectrometry-based imaging. Nine human muscle biopsies from DMD patients and nine muscle biopsies from control individuals were subjected to untargeted MSI using matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry. Both univariate and pattern recognition techniques have been used for data analysis. This study revealed significant changes in 34 keys metabolites. Seven metabolites were decreased in the Duchenne biopsies compared to control biopsies including adenosine triphosphate, and glycerophosphocholine. The other 27 metabolites were increased in the Duchenne biopsies, including sphingomyelin, phosphatidylcholines, phosphatidic acids and phosphatidylserines. Most of these dysregulated metabolites are tightly related to energy and phospholipid metabolism. This study revealed a deep metabolic remodelling in phospholipids and energy metabolism in DMD. This systems-based approach enabled exploring the metabolism in DMD in an unprecedented holistic and unbiased manner with hypothesis-free strategies.

Ketogenic Diet: Impact on Cellular Lipids in Hippocampal Murine Neurons.

BACKGROUND: The mechanism of action of the ketogenic diet (KD), an effective treatment for pharmacotherapy refractory epilepsy, is not fully elucidated. The present study examined the effects of two metabolites accumulating under KD-beta-hydroxybutyrate (ßHB) and decanoic acid (C10) in hippocampal murine (HT22) neurons. METHODS: A mouse HT22 hippocampal neuronal cell line was used in the present study. Cellular lipids were analyzed in cell cultures incubated with high (standard) versus low glucose supplemented with ßHB or C10. Cellular cholesterol was analyzed using HPLC, while phospholipids and sphingomyelin (SM) were analyzed using HPTLC. RESULTS: HT22 cells showed higher cholesterol, but lower SM levels in the low glucose group without supplements as compared to the high glucose groups. While cellular cholesterol was reduced in both ßHB- and C10-incubated cells, phospholipids were significantly higher in C10-incubated neurons. Ratios of individual phospholipids to cholesterol were significantly higher in ßHB- and C10-incubated neurons as compared to controls. CONCLUSION: Changes in the ratios of individual phospholipids to cholesterol in HT22 neurons suggest a possible alteration in the composition of the plasma membrane and organelle membranes, which may provide insight into the working mechanism of KD metabolites ßHB and C10.

Cicer arietinum L. Sprouts' Influence on Mineralization of Saos-2 and Migration of MCF-7 Cells.

In the present study, we investigated the biological activity of four extracts obtained from Cicer arietinum L. sprouts. The fermentation of the sprouts with Lactobacillus casei and their incubation with ß-glucosidase elevated the concentrations of isoflavonoids, especially coumestrol, formononetin and biochanin A. To study the biological activity of C. arietinum, the human osteosarcoma Saos-2 and human breast cancer MCF-7 cell lines were used. The extracts obtained from fermented sprouts exhibited the strongest ability to decrease intracellular oxidative stress in both types of cells. They augmented mineralization and alkaline phosphatase activity in Saos-2 cells, as well as diminished the secretion of interleukin-6 and tumor necrosis factor α. Simultaneously, the extracts, at the same doses, inhibited the migration of MCF-7 cells. On the other hand, elevated concentrations of C. arietinum induced apoptosis in estrogen-dependent MCF-7 cells, while lower doses stimulated cell proliferation. These results are important for carefully considering the use of fermented C. arietinum sprouts as a dietary supplement component for the prevention of osteoporosis.

Decreased R:FR Ratio in Incident White Light Affects the Composition of Barley Leaf Lipidome and Freezing Tolerance in a Temperature-Dependent Manner.

In cereals, C-repeat binding factor genes have been defined as key components of the light quality-dependent regulation of frost tolerance by integrating phytochrome-mediated light and temperature signals. This study elucidates the differences in the lipid composition of barley leaves illuminated with white light or white light supplemented with far-red light at 5 or 15 °C. According to LC-MS analysis, far-red light supplementation increased the amount of monogalactosyldiacylglycerol species 36:6, 36:5, and 36:4 after 1 day at 5 °C, and 10 days at 15 °C resulted in a perturbed content of 38:6 species. Changes were observed in the levels of phosphatidylethanolamine, and phosphatidylserine under white light supplemented with far-red light illumination at 15 °C, whereas robust changes were observed in the amount of several phosphatidylserine species at 5 °C. At 15 °C, the amount of some phosphatidylglycerol species increased as a result of white light supplemented with far-red light illumination after 1 day. The ceramide (42:2)-3 content increased regardless of the temperature. The double-bond index of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine ceramide together with total double-bond index changed when the plant was grown at 15 °C as a function of white light supplemented with far-red light. white light supplemented with far-red light increased the monogalactosyldiacylglycerol/diacylglycerol ratio as well. The gene expression changes are well correlated with the alterations in the lipidome.

In Silico, In Vitro, and In Vivo Antitumor and Anti-Inflammatory Evaluation of a Standardized Alkaloid-Enriched Fraction Obtained from Boehmeria caudata Sw. Aerial Parts.

In the context of the cancer-inflammation relationship and the use of natural products as potential antitumor and anti-inflammatory agents, the alkaloid-enriched fraction of Boehmeriacaudata (BcAEF) aerial parts was evaluated. In vitro antiproliferative studies with human tumor cell lines showed high activity at low concentrations. Further investigation on NCI-H460 cells showed an irreversible effect on cell proliferation, with cell cycle arrest at G2/M phase and programmed cell death induction. Molecular docking studies of four alkaloids identified in BcAEF with colchicine's binding site on ß-tubulin were performed, suggesting (-)-C (15R)-hydroxycryptopleurine as the main inductor of the observed mitotic death. In vivo studies showed that BcAEF was able to reduce Ehrlich tumor volume progression by 30 to 40%. Checking myeloperoxidase activity, BcAEF reduced neutrophils migration towards the tumor. The in vivo anti-inflammatory activity was evaluated by chemically induced edema models. In croton oil-induced ear edema and carrageenan (CG)-induced paw edema models, BcAEF reduced edema around 70 to 80% together with inhibition of activation and/or migration of neutrophils to the inflammatory area. All together the results presented herein show BcAEF as a potent antitumor agent combining antiproliferative and anti-inflammatory properties, which could be further explored in (pre)clinical studies.

Biotherapy of Brain Tumors with Phosphatidylserine-Targeted Radioiodinated SapC-DOPS Nanovesicles.

Glioblastoma multiforme (GBM), a common type of brain cancer, has a very poor prognosis. In general, viable GBM cells exhibit elevated phosphatidylserine (PS) on their membrane surface compared to healthy cells. We have developed a drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), that selectively targets cancer cells by honing in on this surface PS. To examine whether SapC-DOPS, a stable, blood-brain barrier-penetrable nanovesicle, could be an effective delivery system for precise targeted therapy of radiation, we iodinated several carbocyanine-based fluorescent reporters with either stable iodine (127I) or radioactive isotopes (125I and 131I). While all of the compounds, when incorporated into the SapC-DOPS delivery system, were taken up by human GBM cell lines, we chose the two that best accumulated in the cells (DiI (22,3) and DiD (16,16)). Pharmacokinetics were conducted with 125I-labeled compounds and indicated that DiI (22,3)-SapC-DOPS had a time to peak in the blood of 0.66 h and an elimination half-life of 8.4 h. These values were 4 h and 11.5 h, respectively, for DiD (16,16)-SapC-DOPS. Adult nude mice with GBM cells implanted in their brains were treated with 131I-DID (16,16)-SapC-DOPS. Mice receiving the radionuclide survived nearly 50% longer than the control groups. These data suggest a potential novel, personalized treatment for a devastating brain disease.

Protective effect of vitamin C and lycopene on the in vitro fertilization capacity of sex-sorted bull sperm by inhibiting the oxidative stress.

The fertilization capacity of sex-sorted sperms is seriously decreased, which inhibits its wide application. However, little information is still available about the effect of vitamin C (VC) and lycopene (Lyc) on the fertilization capacity of sex-sorted bull sperm. In this study, the washing medium and fertilization medium of sex-sorted sperm from three bull individuals were supplemented with different concentrations of VC (0, 1 × 10-3 , 1 × 10-4 , 1 × 10-5 , 1 × 10-6  M) or Lyc (0, 1 × 10-4 , 1 × 10-5 , 1 × 10-6 , 1 × 10-7 ). After washing twice and incubation for 1.5 hr, the malondialdehyde (MDA) level, phosphatidylserine (PS) translocation, membrane potential (Δψm) and IVF (in vitro fertilization) ability of sex-sorted sperm were investigated. For the sex-sorted sperm of bulls A, B and C, 1 × 10-3  M VC or 1 × 10-4  M Lyc treatment significantly decreased their MDA levels and PS translocation and increased their Δψm levels and cleavage rates after IVF. When blastocysts were concerned, 1 × 10-4  M Lyc significantly improved the blastocyst rates and their IFN-tau expression of bulls A and C. In conclusion, supplementation of 1 × 10-3  M VC or 1 × 10-4  M Lyc in washing and fertilization medium contributed greatly to improving the fertilization capacity of sex-sorted bull sperm during IVF procedure.

Recovery of brain DHA-containing phosphatidylserine and ethanolamine plasmalogen after dietary DHA-enriched phosphatidylcholine and phosphatidylserine in SAMP8 mice fed with high-fat diet.

BACKGROUND: Glycerophospholipids were the main components of cerebral cortex lipids, and there was a close association between lipid homeostasis and human health. It has been reported that dietary DHA-enriched phosphatidylcholine (DHA-PC) and phosphatidylserine (DHA-PS) could improve brain function. However, it was unclear that whether supplementation of DHA-PC and DHA-PS could change lipid profiles in the brain of dementia animals. METHODS: SAMP8 mice was fed with different diet patterns for 2 months, including high-fat diet and low-fat diet. After intervention with DHA-PC and DHA-PS for another 2 months, the lipid profile in cerebral cortex was determined by lipidomics in dementia mice. RESULTS: High-fat diet could significantly decrease the levels of DHA-containing PS/pPE, DPA-containing PS, and AA-containing PE, which might exhibit the potential of lipid biomarkers for the prevention and diagnosis of AD. Notably, DHA-PC and DHA-PS remarkably recovered the lipid homeostasis in dementia mice. These might provide a potential novel therapy strategy and direction of dietary intervention for patients with cognitive decline. CONCLUSIONS: DHA-PC and DHA-PS could recover the content of brain DHA-containing PS and pPE in SAMP8 mice fed with high-fat diet.

Bifidobacterium longum subsp. longum Remodeled Roseburia and Phosphatidylserine Levels and Ameliorated Intestinal Disorders and liver Metabolic Abnormalities Induced by High-Fat Diet.

Bifidobacterium longum is considered as a potential supplement in antiobesity treatment; however, the underlying molecular mechanism has rarely been studied. To understand the contributions of B. longum subsp. longum (BL21) in the prevention of obesity, we investigated alterations in the liver metabonomic phenotype and gut microbiota by ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and 16S ribosomal RNA gene sequencing in C57BL/6J male mice orally administered with BL21 for 8 weeks [high-fat diet (HFD)]. BL21 at 1 × 109 CFU·day-1 per mouse reduced the weight of mice by 16.9% relative to that of the mice fed with HFD and significantly lowered the serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol. BL21 also ameliorated fat vacuolization in liver cells and epididymal fat accumulation. BL21 also lowered the Firmicutes/Bacteroidetes ratio, regulated liver remodeling in glycerophospholipids, and alleviated the levels of d-tryptophan. A positive correlation between the butyrate-producing strain Roseburia and the cell membrane component phosphatidylserine was found for the first time. Thus, BL21 can potentially prevent mice from being obese by rebalancing the gut microbiota and glycerophospholipid metabolism. BL21 can be a promising dietary supplement for weight control.

Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity.

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.

Interactions between neutrophil extracellular traps and activated platelets enhance procoagulant activity in acute stroke patients with ICA occlusion.

BACKGROUND: The role of neutrophil extracellular traps (NETs) in procoagulant activity (PCA) in stroke patients caused by thromboembolic occlusion of the internal carotid artery (ICA) remains unclear. Our objectives were to evaluate the critical role of NETs in the induction of hypercoagulability in stroke and to identify the functional significance of NETs during atherothrombosis. METHODS: The levels of NETs, activated platelets (PLTs), and PLT-derived microparticles (PMPs) were detected in the plasma of 55 stroke patients and 35 healthy controls. NET formation and thrombi were analysed using immunofluorescence. Exposed phosphatidylserine (PS) was evaluated with flow cytometry and confocal microscopy. PCA was analysed using purified coagulation complex, thrombin, and fibrin formation assays. FINDINGS: The plasma levels of NETs, activated PLTs, and PMP markers in the carotid lesion site (CLS) were significantly higher than those in the aortic blood. NETs were decorated with PS in thrombi and the CLS plasma of ICA occlusion patients. Notably, the complementary roles of CLS plasma and thrombin-activated PLTs were required for NET formation and subsequent PS exposure. PS-bearing NETs provided functional platforms for PMPs and coagulation factor deposition and thus increased thrombin and fibrin formation. DNase I and lactadherin markedly inhibited these effects. In addition, NETs were cytotoxic to endothelial cells, converting these cells to a procoagulant phenotype. Sivelestat, anti-MMP9 antibody, and activated protein C (APC) blocked this cytotoxicity by 25%, 39%, or 52%, respectively. INTERPRETATION: NETs played a pivotal role in the hypercoagulability of stroke patients. Strategies that prevent NET formation may offer a potential therapeutic strategy for thromboembolism interventions. FUNDING: This study was supported by grants from the National Natural Science Foundation of China (61575058, 81873433 and 81670128) and Graduate Innovation Fund of Harbin Medical University (YJSKYCX2018-58HYD).