Blockade of CD300A enhances the ability of human NK cells to lyse hematologic malignancies

Objective: The human cluster of differentiation(CD)300A, a type-I transmembrane protein with immunoreceptor tyrosine-based inhibitory motifs, was investigated as a potential immune checkpoint for human natural killer(NK) cells targeting hematologic malignancies(HMs).Methods: We implemented a stimulation system involving the CD300A ligand, phosphatidylserine(PS), exposed to the outer surface of malignant cells. Additionally, we utilized CD300A overexpression, a CD300A blocking system, and a xenotransplantation model to evaluate the impact of CD300A on NK cell efficacy against HMs in in vitro and in vivo settings. Furthermore, we explored the association between CD300A and HM progression in patients.Results: Our findings indicated that PS hampers the function of NK cells. Increased CD300A expression inhibited HM lysis by NK cells. CD300A overexpression shortened the survival of HM-xenografted mice by impairing transplanted NK cells. Blocking PS–CD300A signals with antibodies significantly amplified the expression of lysis function-related proteins and effector cytokines in NK cells, thereby augmenting the ability to lyse HMs. Clinically, heightened CD300A expression correlated with shorter survival and an “exhausted” phenotype of intratumoral NK cells in patients with HMs or solid tumors.Conclusions: These results propose CD300A as a potential target for invigorating NK cell-based treatments against HMs.

Dietary Lipid Intervention in the Prevention of Brain Aging

As people live longer,the burden of aging-related brain diseases,especially dementia,is increasing.Brain aging increases the risk of cognitive impairment,which manifests as a progressive loss of neuron function caused by the impairment of synaptic plasticity via disrupting lipid homeostasis.Therefore,supplemental dietary lipids have the potential to prevent brain aging.This review summarizes the important roles of dietary lipids in brain function from both structure and mechanism perspectives.Epidemiological and animal studies have provided evidence of the functions of polyunsaturated fatty acids(PUFAs)in brain health.The results of interventions indicate that phospholipids—including phosphatidylcholine,phosphatidylserine,and plasmalogen—are efficient in alleviating cognitive impairment during aging,with plasmalogen exhibiting higher efficacy than phosphatidylserine.Plasmalogen is a recognized nutrient used in clinical trials due to its special vinyl ether bonds and abundance in the postsynaptic membrane of neurons.Future research should determine the dose-dependent effects of plasmalogen in alleviating brain-aging diseases and should develop extraction and storage procedures for its clinical application.

Aptasensing biosynthesized phosphatidylserine with a AuNPs nanozyme-based colorimetric aptasensor

Sensitive monitoring of the target products during the biosynthesis process is crucial,and facile analytical approaches are urgently needed.Herein,phosphatidylserine(PS)was chosen as the model target,a colorimetric aptasensor was developed for the rapid quantitation in biosynthesis samples.A chimeric aptamer was constructed with two homogeneous original PS aptamers.Specific recognition between the chimeric aptamer and PS results in the desorption of aptamer from the surface of the AuNPs nanozyme,and the peroxidase-like enzymatic activity of the AuNPs nanozyme was weakened in a relationship with the different concentrations.The developed aptasensor performed well when applied for analyzing PS in biosynthesis samples.The aptasensor offers good sensitivity and selectivity,under optimal conditions,achieving monitoring and quantitation of PS in the range of 2.5-80.0μmol/L,with a limit of detection at 536.2 nmol/L.Moreover,the aptasensor provides good accuracy,with comparison rates of 98.17%-106.40%,when compared with the HPLC-ELSD.This study provides a good reference for monitoring other biosynthesized products and promoting the development of aptamers and aptasensors in real-world applications.

Phosphatidylserine:An overview on functionality,processing techniques,patents,and prospects

Phosphatidylserine(PS)is the part of cell structure in the body and has many beneficial functions especially in brain-related aging diseases.Although daily foods can provide PS to human body,the amount is very limited due to its poverty in most foods.To overcome the issue,numerous studies based on PS have been reported to develop PS-related supplements.In this review,PS was comprehensively and critically reviewed from the view of resources,functions,processing techniques,patents,and prospects.For resources,animal,plant,and microorganism origins were all covered with their differences in composition profiles.For functions,benefits regarding memory,cognitive enhancement,exercise performance,reducing Alzheimer’s disease,and attention-deficit hyperactivity disorder symptoms were covered as well as the functional differences among animal-,plant-,and microorganism-based PS-related supplements.For processing techniques,traditional extracting methods from animal,plant,and microorganism tissues were comparatively discussed with enzymatic synthesis based on different reaction systems.Finally,patents of PS-related supplements were evaluated as well as their applications.This review could provide scientific and valuable support for PS industry.

Early apoptosis in intestinal and diffuse gastric carcinomas

INTRODUCTIONApoptosis,described by Kerr in 1972,plays a keyrole in all types of regulated cellular processes inmulticellular,organisms.It is defined as amorphologic change,including fragmentation of theDNA,cell shrinkage,dilation of the endoplasmaticreticulum,cell fragmentation and formation

Phosphatidylserine improves axonal transport by inhibition of HDAC and has potential in treatment of neurodegenerative diseases

Familial dysautonomia （FD） is a rare children neurodegenerative disease caused due to a point mutation in the IKBKAP gene that results in decreased IKK complex-associated protein （IKAP） protein production. The disease affects mostly the dorsal root ganglion （DRG） and the sympathetic ganglion. Recently, we found that the molecular mechanisms underlying neurodegeneration in FD patients are defects in axonal transport of nerve growth factors and microtubule stability in the DRG. Neurons are highly polarized cells with very long axons. In order to survive and maintain proper function, neurons depend on transport of proteins and other cellular components from the neuronal body along the axons. We further demonstrated that IKAP is necessary for axon maintenance and showed that phosphatidylserine acts as an HDAC6 inhib- itor to rescue neuronal function in FD cells. In this review, we will highlight our latest research findings.

Bridging the gap:axonal fusion drives rapid functional recovery of the nervous system

Injuries to the central or peripheral nervous system frequently cause long-term disabilities because damaged neurons are unable to efficiently self-repair.This inherent deficiency necessitates the need for new treatment options aimed at restoring lost function to patients.Compared to humans,a number of species possess far greater regenerative capabilities,and can therefore provide important insights into how our own nervous systems can be repaired.In particular,several invertebrate species have been shown to rapidly initiate regeneration post-injury,allowing separated axon segments to re-join.This process,known as axonal fusion,represents a highly efficient repair mechanism as a regrowing axon needs to only bridge the site of damage and fuse with its separated counterpart in order to re-establish its original structure.Our recent findings in the nematode Caenorhabditis elegans have expanded the promise of axonal fusion by demonstrating that it can restore complete function to damaged neurons.Moreover,we revealed the importance of injury-induced changes in the composition of the axonal membrane for mediating axonal fusion,and discovered that the level of axonal fusion can be enhanced by promoting a neuron＇s intrinsic growth potential.A complete understanding of the molecular mechanisms controlling axonal fusion may permit similar approaches to be applied in a clinical setting.

SYNGR4 and PLEKHB1 deregulation in motor neurons of amyotrophic lateral sclerosis models: potential contributions to pathobiology

Amyotrophic lateral sclerosis is the most common adult-onset neurodegenerative disease affecting motor neurons. Its defining feature is progressive loss of motor neuron function in the cortex, brainstem, and spinal cord, leading to paralysis and death. Despite major advances in identifying genes that can cause disease when mutated and model the disease in animals and cellular models, it still remains unclear why motor symptoms suddenly appear after a long pre-symptomatic phase of apparently normal function. One hypothesis is that age-related deregulation of specific proteins within key cell types, especially motor neurons themselves, initiates disease symptom appearance and may also drive progressive degeneration. Genome-wide in vivo cell-type-specific screening tools are enabling identification of candidates for such proteins. In this minireview, we first briefly discuss the methodology used in a recent study that applied a motor neuron-specific RNASeq screening approach to a standard model of TAR DNA-binding protein-43(TDP-43)-driven amyotrophic lateral sclerosis. A key finding of this study is that synaptogyrin-4 and pleckstrin homology domain-containing family B member 1 are also deregulated at the protein level within motor neurons of two unrelated mouse models of mutant TDP-43 driven amyotrophic lateral sclerosis. Guided by what is known about molecular and cellular functions of these proteins and their orthologs, we outline here specific hypotheses for how changes in their levels might potentially alter cellular physiology of motor neurons and detrimentally affect motor neuron function. Where possible, we also discuss how this information could potentially be used in a translational context to develop new therapeutic strategies for this currently incurable, devastating disease.

Down Regulation of MyD88 in Macrophages Treated with Liposomes Composed of Phosphatidylserine

We have recently demonstrated that liposomes composed of phosphatidylserine (PS-liposomes) suppressed nitric oxide and inflammatory cytokine productions following LPS stimulation in macrophages. In this study, we examined the effect of PS-liposomes on expressions of TLR-4 and MyD88, which are essential for the signal transduction in LPS stimulation. Expression of MyD88 was suppressed when macrophages were treated with PS-liposomes, but not with liposomes of phosphatidylcholine. No change in TLR-4 expression was observed. MyD88 suppression was restored to the control levels when cells were pre-treated with anti-TGF-β antibody, suggesting that TGF-β plays an important role in down-regulation of MyD88 following PS-liposome treatment.

Chemokine SR-PSOX/CXCL16 expression in peripheral blood of patients with acute coronary syndrome

Background Scavenger receptor that binds phosphatidylserine and oxidized lipoprotein/CXC chemokine ligand 16 （SR-PSOX/CXCL16） promotes foam cell formation through the tumor necrosis factor （TNF）-α mediated mechanism. Because chemokine CXCL16 could be expressed in atherosclerotic lesions and induce smooth muscle cell （SMC） proliferation, we＇presume that the monocyte SR-PSOX/CXCL16 detection in the patients＇ peripheral blood will be important for early diagnosis and prognosis of atherosclerosis （AS）. Methods Enrolled in this study were 40 patients with acute coronary syndrome （ACS）, including 20 patients with acute myocardial infarction （AMI） and 20 patients with unstable angina pectoris （UAP）, and 20 normal controls. Monocytes in the peripheral blood were isolated, and the changes of expression of CXCL16/SR-PSOX mRNA were compared using reverse transcription-polymerase chain reaction （RT-PCR）, with β-actin as internal control. We compared the expression of CXCL16/SR-PSOX in the ACS subgroups, using Western-blot to analyze protein expression levels. Tissue sections were made from biopsy specimens taken from patients with infective endocarditis, liver cirrhosis, and lung cancer as well as normal controls. And the expression of CXCL16/SR- PSOX was analyzed with a confocal microscope. Results The expression of CXCL16/SR-PSOX mRNA and protein in the monocytes of peripheral blood was significantly higher in ACS patients than in normal controls （P〈0.05）; however, there was no significant difference in CXCL16/SR-PSOX expression between UAP group and AMI group （P〉0.05）. Immunofluorescence showed that there were low expression of SR-PSOX in normal vascular endothelial cells and enhanced expression in every layer of the infected vessels, while spreading from endothelial cells to surrounding tissues as infection worsens. Confocal microscopy showed that the expression of SR-PSOX was enhanced in the infiltrated lymphocytes in liver cirrhosis, and that the expression level was proportionate to the degree of inflammation in the portal hepatis and folia. Conclusions The expression of CXCL16/SR-PSOX in the monocytes of peripheral blood was significantly higher in ACS patients than in the controls. CXCL16/SR-PSOX-mediated inflammation may contribute to the pathogenesis of ACS, and CXCL16 may play an important role in the pathogenesis and development of AS in humans.

Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis

Background:Understanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies.Tumor cells often undergo spontaneous apoptotic cell death in tumor microen-vironment,these apoptotic cells are histologically co-localized with immunosup-pressive macrophages.However,the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood.In this study,we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved.Methods:Apoptotic cells and macrophages in tumors were detected by immunohistochemical staining.Morphological analysis was performed with Giemsa staining.Lipids generated from apoptotic cells were detected by liq-uid chromatography-mass spectrometry.Phosphatidylserine-containing lipo-somes were prepared to mimic apoptotic cells.The expression of protein was determined by real-time PCR,immunohistochemistry enzyme-linked immunosorbent assay and Western blotting.Mouse malignant ascites and subcu-taneous tumor models were designed for in vivo analysis.Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies.Results:The location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas.Phosphatidylserine,a lipid molecule generated in apoptotic cells,induced polarization and accumulation of M2-like macrophages in vivo and in vitro.Moreover,sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcuta-neous tumor models.Further analyses suggested that phosphoserine induced a M2-like phenotype in macrophages,which was related to the activation of phosphoserine receptors including T-cell immunoglobin mucin 4(TIM4)and the FAK-SRC-STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain-containing protein 3(JMJD3).Adminis-tration of specific inhibitors of these pathways could reduce tumor progression.Conclusions:This study suggest that apoptotic cell-generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors,and the related pathways might be potential therapeutic targets for cancer therapy.

Surfactant protein A (SP-A) binds to phosphatidylserine and competes with annexin V binding on late apoptotic cells

The role of surfactant protein A(SP-A)in the recognition and clearance of apoptotic cells is well established,but to date,it is still not clear which surface molecules of apoptotic cells are involved in the process.Here we present evidence that phosphatidylserine(PS)is a relevant binding molecule for human SP-A.The binding is Ca^(2+)-dependent and is not inhibited by mannose,suggesting that the sugar-binding site of the carbohydrate recognition domain(CRD)of SP-A is not involved.Flow cytometry studies on apoptotic Jurkat cells revealed apparent inhibition of annexin V binding by increasing concentrations of SP-A in late apoptotic but not early apoptotic cells,and this was consistent for Jurkat cells and neutrophils.Supporting these data,confocal microscopy results show a co-localisation of annexin V and SP-A in late apoptotic but not early apoptotic cells.However,we cannot conclude that this inhibition is exclusively due to the binding of SP-A to PS on the cell surface,as annexin V is not wholly specific for PS and SP-A also interacts with other phospholipids that might become exposed on the apoptotic cell surface.

Increased procoagulant activity of red blood cells in the presence of cisplatin

Background Cisplatin based chemotherapy is a well recognized risk factor for coagulation disorders and thrombosis. The pathophysiological mechanisms by which cisplatin promote thrombosis are not well understood. Methods Red blood cells （RBCs） were separated from peripheral blood of patients with breast cancer （n=10） and healthy adults （n=6） and treated with cisplatin. Coagulation time of RBCs was assessed by one step recalcification time and the productions of thrombin, intrinsic and extrinsic factor Xa were measured in the presence or absence of various concentrations of lactadherin. Exposed phosphatidylserine was stained with lactadherin and observed by confocal microscopy and flow cytometry. Results Neither fresh RBCs nor RBCs treated without cisplatin had potent procoagulant activity. Cisplatin treatment increased procoagulant activity of RBCs in a cell number- and concentration-dependent manner. Exposed phosphatidylserine was stained with lactadherin and after cisplatin treatment, strong fluorescence was revealed by confocal microscopy. Lactadherin bound RBCs from patients with breast cancer increased from （1.9＋0.5）% on control RBCs to （68.0±3.5）% on RBCs treated with 10umol/L cisplatin for 24 hours. Conclusions Cisplatin treatment increases procoagulant activity of RBCs, which have a strong association with exposure of phosphatidylserine. The increased procoagulant activity may contribute to the pathogenesis of thrombophilia during cisplatin based chemotherapy in breast cancer patients.

MACS-annexin V cell sorting of semen samples with high TUNEL values decreases the concentration of cells with abnormal chromosomal content:a pilot study

We question whether,in men with an abnormal rate of sperm DNA fragmentation,the magnetic-activated cell sorting(MACS)could select spermatozoa with lower rates of DNA fragmentation as well as spermatozoa with unbalanced chromosome content.Cryopreserved spermatozoa from six males were separated into nonapoptotic and apoptotic populations.We determined the percentages of spermatozoa with(i)externalization of phosphatidylserine(EPS)by annexin V-Fluorescein isothiocyanate(FITC)labeling,(ii)DNA fragmentation by TdT-mediated-dUTP nick-end labeling(TUNEL),and(iii)numerical abnormalities for chromosomes X,Y,13,18,and 21 by fluorescence in situ hybridization(FISH),on the whole ejaculate and selected spermatozoa in the same patient.Compared to the nonapoptotic fraction,the apoptotic fraction statistically showed a higher number of spermatozoa with EPS,with DNA fragmentation,and with numerical chromosomal abnormalities.Compared to the whole ejaculate,we found a significant decrease in the percentage of spermatozoa with EPS and decrease tendencies of the DNA fragmentation rate and the sum of disomy levels in the nonapoptotic fraction.Conversely,we observed statistically significant higher rates of these three parameters in the apoptotic fraction.MACS may help to select spermatozoa with lower rates of DNA fragmentation and unbalanced chromosome content in men with abnormal rates of sperm DNA fragmentation.

ADAM10 sheddase activation is controlled by cell membrane asymmetry

Dysregulation of the disintegrin-metalloproteinase ADAM10 may contribute to the development of diseases including tumorigenesis and Alzheimer's disease.The mechanisms underlying ADAM10 sheddase activation are incompletely understood.Here,we show that transient exposure of the negatively charged phospholipid phosphatidylserine(PS)is necessarily required.The soluble PS headgroup was found to act as competitive inhibitor of substrate cleavage.Overexpression of the Ca2+-dependent phospholipid scramblase Anoctamin-6(AN06)led to increased PS externalization and substrate release.Transfection with a constitutively active form of AN06 resulted in maximum sheddase activity in the absence of any stimulus.Calcium-dependent ADAM10 activation could not be induced in lymphocytes of patients with Scott syndrome harbouring a missense mutation in AN06.A putative PS-binding motif was identified in the conserved stalk region.Replacement of this motif resulted in strong reduction of sheddase activity.In conjunction with the recently described 3D structure of the ADAM10 extracellular domain,a model is advanced to explain how surface-exposed PS triggers ADAM 10 sheddase function.

Oviductal epithelial cells selected boar sperm according to their functional characteristics

The interaction of oviductal epithelial cells （OECs） with the spermatozoa has beneficial effects on the sperm functions. The aim of this study is to evaluate the in vitro fertilizing capacity of incubating spermatozoa previously selected by density gradient in OEC and determinate some sperm characteristics that could explain the results obtained. In this study, we assessed in vitro fertilization （IVF）, tyrosine phosphorylation, phosphatidylserine translocation, nuclear DNA fragmentation, and chromatin decondensation. Three experimental sperm groups, previously selected by Percoll gradient, were established according to the origin of the sperm used for IVF： （i） W30 group： spermatozoa were incubated with oocytes in the absence of OEC; （ii） NB group： after sperm incubation in OEC, the unbound spermatozoa were incubated with oocytes, in the absence of OEC; and （iii） B group： after sperm incubation with OEC, the bound spermatozoa were incubated with oocytes in the OEC plates. The results showed that sperm from the NB group led to a lower IVF yield, accompanied by low penetration rates （NB： 19.6%, B： 94.9%, and W30： 62.9%; P 〈 0.001） and problems of nuclear decondensation. Moreover, higher levels of tyrosine phosphorylation were observed in the NB group compared with the W30 and B groups （NB： 58.7%, B： 2.5%, and W30： 4.5%; P 〈 0.01）. A similar trend was observed in phosphatidylserine translocation （NB： 93.7%, B. 5.7%, and W30： 44.2%; P 〈 0.01）. These results demonstrate that the OEC exerts a rigorous degree of sperm selection, even within an already highly selected population of spermatozoa, and can capture the best functional spermatozoa for fertilization.

Recombinant protein diannexin prevents preeclampsia-like symptoms in a pregnant mouse model via reducing the release of microparticles

Preeclampsia(PE)is characterized by placenta-mediated pregnancy complication.The only effective treatment for PE is the delivery of the placenta.However,this treatment may cause preterm birth and neonatal death.Therefore,preventing PE is needed.The mechanism of PE involves abnormal placentation,which leads to the release of anti-angiogenic and inflammatory mediators into maternal circulation.These mediators contribute to systemic vascular dysfunction,inflammatory responses,and excessive thrombin generation.Microparticles(MPs)are reportedly involved in PE by promoting the thromboinflammatory response.This study describes a strategy to prevent PE by reducing MP release using the recombinant protein,diannexin.Results showed that the patients with PE had elevated MP number and procoagulant activity and increased NLRP3 inflammasome activation.Additionally,diannexin remarkably reduced the release of MPs from activated cells by binding to phosphatidylserine exposed on the surface of activated cells.Moreover,in vivo results showed that diannexin could prevent PE-like symptoms by decreasing MPs and NLRP3 inflammasome activation in pregnant mice.Furthermore,diannexin effectively inhibited trophoblast cell activation and NLRP3 inflammasome activation in vitro.These findings suggested that diannexin inhibited MP release and might be an effective therapeutic strategy for preventing PE.

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 NKcell 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.

Peptide ligand-SiO_(2) microspheres with specific affinity for phosphatidylserine as a new strategy to isolate exosomes and application in proteomics to differentiate hepatic cancer

Exosomes are membrane bound extracellular vesicles that play an important role in many biological processes.While they have great application value,exosome isolation is still considered a major scientific challenge.In the present study,a novel separation strategy for exosomes is proposed based on the specific interaction between immobilized peptide ligands and phosphatidylserine moieties which are highly abundant on the surface of exosomes.With the new affinity method,intact model exosomes can be recovered with a high yield in a short processing time.The purity of exosome samples enriched from serum by the affinity method is far higher than that isolated by ultrafiltration,and similar to that obtained by density gradient centrifugation and ultracentrifugation.Moreover,the variety of contaminants co-isolated by the affinity method is relatively low due to its specific separation principle.Proteomics analysis of exosomes isolated by the affinity method from the serum of healthy,hepatocellular carcinoma patients,and intrahepatic cholangiocarcinoma patients was performed to prove the applicability of this method.In conclusion,our novel strategy shows characteristics of easy preparation,high specificity,and cost-effectiveness,and provides a promising approach for exosome isolation which should have wide applications.