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Ischemia-reperfusion-induced acute kidney injury (IR-AKI) commonly occurs in situations such as hemorrhagic shock, kidney transplantation, and cardiovascular surgery. As one of the significant causes of AKI, IR-AKI is characterized by its high incidence and mortality rates. Currently, effective inflammation control is the key for the treatment of IR-AKI. In this study, we developed an ROS-responsive polymeric prodrugs (Zn-D/DTH) which could target the externalized PS of apoptotic cells, and then responsively released HDM (anti-inflammatory peptides) in the presence of intracellular ROS. Zn-D/DTH effectively ameliorated renal function and mitigated pathological alterations such as the loss of the brush border, tubular dilation, and accumulation of cellular debris within the tubular lumens. Furthermore, Zn-D/DTH greatly reduced the generation of pro-inflammatory factors like IL-6, COX-2, and iNOS in renal tissues, suggesting its protective role largely stems from suppression of the inflammatory response. Additional mechanism exploration revealed that Zn-D/DTH markedly decreased the expression levels of TLR4 and MyD88, as well as the phosphorylation of NF-κB in the damaged kidneys. This, in turn, reduced the number of apoptotic tubular cells and the activity of Caspase 9 and Caspase 3 caused by ischemia-reperfusion. Additionally, Zn-D/DTH treatment showed improvement in the long-term renal damage and fibrosis induced by ischemia-reperfusion. The experimental outcomes indicated that Zn-D/DTH attenuated renal ischemia-reperfusion injury and delayed the transition from acute kidney injury to chronic kidney disease by downregulating the TLR4/MyD88/NF-κB signaling pathway and reducing the expression of apoptotic caspases, thereby inhibiting inflammation and reducing cell apoptosis. Design strategy of Zn-D/DTH and schematic representation of the mode of action proposed for the treatment of IR-AKI. [Display omitted] • A PS-targeting ROS-responsive polymeric prodrug Zn-D/DTH for the treatment of ischemia-reperfusion-induced acute kidney injury. • Zn-D/DTH could effectively ameliorate renal function and mitigate pathological alterations. • Zn-D/DTH reduced pro-inflammatory factors and apoptotic cells via inhibiting TLR4/MyD88/NF-kappaB signaling pathway. • Zn-D/DTH was good at mitigating long-term renal damage and fibrosis triggered by ischemia-reperfusion. [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) has many applications in functional food and pharmaceutical industries because of its critical role in activating important signal transduction pathways and modulating neurotransmitter release and receptor function. Several edible oils have been used to construct oil–water systems for the efficient production of PS under mild conditions by phospholipase D (PLD)‐mediated transphosphatidylation. Toxic organic solvents were completely avoided throughout the process, from manufacturing to encapsulation. Results showed that higher enzyme selectivity and activity were found in the coconut oil–water and olive oil–water systems than in the traditional diethyl ether–water system. The maximum yields of PS in the two oil–water systems were more than 93%, whereas that in the diethyl ether–water system was only 78%. Hence, the coconut oil–water and olive oil–water systems should be the promising medium to produce PS and its microcapsules. Moreover, the enzymatic mechanism of PLD was simulated using molecular dynamics (MD) to understand diffusion channels, competitive bindings, solvent effects, structural changes, system stabilities, and product dissociation from the enzyme. MD results showed that more PLD‐substrate complexes were detected in the oil–water systems than in the diethyl ether–water system. The conformational transition states of the protein between open and closed conformations were directly affected by the solvents used, and structural changes were observed in loop I (Asp309‐Thr336). PLD in oil–water systems can not only maintain the native structure but also broaden the diffusion channels for substrates, which contributes to a higher enzymatic activity. [ABSTRACT FROM AUTHOR]

T-cell immunoglobulin and mucin (TIM) family proteins facilitate the clearance of apoptotic cells, are involved in immune regulation, and promote infection of enveloped viruses. These processes are frequently studied in experimental animals, such as mice or rhesus macaques, but functional differences among the TIM orthologs from these species have not been described. Previously, we reported that while all three human TIM proteins bind phosphatidylserine (PS), only human TIM1 (hTIM1) binds phosphatidylethanolamine (PE), and that this PE-binding ability contributes to both phagocytic clearance of apoptotic cells and viral infection. Here, we show that rhesus macaque TIM1 (rhTIM1) and mouse TIM1 (mTIM1) bind PS but not PE, and that their inability to bind PE makes them less efficient than hTIM1. We also show that alteration of only two residues of mTIM1 or rhTIM1 enables them to bind both PE and PS, and that these PE-binding variants are more efficient at phagocytosis and mediating viral entry. Further, we demonstrate that the mucin domain also contributes to the binding of the virions and apoptotic cells, although it does not directly bind phospholipid. Interestingly, contribution of the hTIM1 mucin domain is more pronounced in the presence of a PE-binding head domain. These results demonstrate that rhTIM1 and mTIM1 are inherently less functional than hTIM1, owing to their inability to bind PE and their less functional mucin domains. They also imply that mouse and macaque models underestimate the activity of hTIM1. IMPORTANCE We previously reported that human T-cell immunoglobulin and mucin protein 1 (TIM1) binds phosphatidylethanolamine (PE) as well as phosphatidylserine (PS), and that PE is exposed on the apoptotic cells and viral envelopes. Moreover, TIM1 recognition of PE contributes to phagocytic clearance of apoptotic cells and virus uptake. Here, we report that unlike human TIM1, murine and rhesus TIM1 orthologs bind only PS, and as a result, their ability to clear apoptotic cells or promote virus infection is less efficient. These findings are significant because they imply that the activity of TIM1 in humans is greater than what the studies conducted in common animal models would indicate. [ABSTRACT FROM AUTHOR]

Chronic nonspecific low back pain (cNLBP) accounts for approximately 90% of low back pain cases, affecting 65–80% of the population and significantly impacting life quality and productivity. This condition also leads to substantial financial burden. Although there have been advancements, a comprehensive understanding of the underlying etiology of cNLBP remains elusive, resulting in less than optimal treatment outcomes. This study aimed to examine the correlation between lipid variations and the development of cNLBP. The cohort consisted of 26 healthy volunteers (HV group) and 30 cNLBP patients, with an assessment of metabolites and lipid composition in both groups. Metabolomic results revealed significant alterations in lipid-associated metabolites between the HV and cNLBP groups. Subsequent lipid analysis revealed that monoacylglycerols (MAGs) increased approximately 1.2-fold (p = 0.016), diacylglycerols (DAGs) increased approximately 1.4-fold (p = 0.0003), and phosphatidylserine (PS) increased approximately 1.4-fold (p = 0.011). In contrast, triacylglycerol (TAG) decreased to about 0.7-fold (p = 0.035) in the cNLBP group compared to the HV group. The contrasting trends in MAG/DAG and TAG levels indicated that the imbalance between MAG/DAG and TAG may have an impact on the development of cNLBP. This study has provided new insights into the relationship between the progression of cNLBP and specific lipids, suggesting that these lipids could serve as therapeutic targets for cNLBP. [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) has significant applications in various sectors, such as the medical and food industries. However, its production relies heavily on phospholipase D (PLD), a crucial tool that is hindered by issues like poor stability and irrecoverability. Immobilization presents itself as an effective solution to overcome these limitations. In this study, magnetic graphene oxide (MGO) modified with an amino (NH2) group was synthesized and utilized for PLD immobilization. The activity of the immobilized PLD (MGO-PLD) reached 3062 U/gMGO, with a specific activity of 33.9 U/mgPLD, virtually identical to that of the free PLD. MGO-PLD was utilized to synthesize PS efficiently in a biphasic system. Under optimal conditions, the PS yield reached 18.66 g/L, with a conversion rate of 92.8% and a productivity of 3.11 g/L/h. Notably, MGO-PLD retained an impressive PS conversion rate of 77.4% even after seven repetitive usages. Moreover, MGO-PLD displayed enhanced thermal and pH resistance properties compared to free PLD, alongside augmented storage stability. After an 8-week preservation at 4 °C, its residual activity was maintained at 76.3%. This study provides a sustainable and highly efficient pathway for the biocatalytic synthesis of PS. [ABSTRACT FROM AUTHOR]

Over the past 40 years, research has heavily emphasized stroke treatments that directly target ischemic cascades after stroke onset. Much attention has focused on studying neuroprotective drugs targeting one aspect of the ischemic cascade. However, the single-target therapeutic approach resulted in minimal clinical benefit and poor outcomes in patients. Considering the ischemic cascade is a multifaceted and complex pathophysiological process with many interrelated pathways, the spotlight is now shifting towards the development of neuroprotective drugs that affect multiple aspects of the ischemic cascade. Phosphatidylserine (PS), known as the "eat-me" signal, is a promising candidate. PS is involved in many pathophysiological changes in the central nervous system after stroke onset, including apoptosis, inflammation, coagulation, and neuronal regeneration. Moreover, PS might also exert various roles in different phases after stroke onset. In this review, we describe the synthesis, regulation, and function of PS under physiological conditions. Furthermore, we also summarize the different roles of PS after stroke onset. More importantly, we also discuss several treatment strategies that target PS. We aim to advocate a novel stroke care strategy by targeting PS through a translational perspective. [ABSTRACT FROM AUTHOR]

BACKGROUND: Lack of n‐3 polyunsaturated fatty acids during the period of maternity drastically lowers the docosahexaenoic acid (DHA) level in the brain of offspring and studies have demonstrated that different molecular forms of DHA are beneficial to brain development. The aim of this study was to investigate the effect of short‐term supplementation with DHA‐enriched phosphatidylserine (PS) and phosphatidylcholine (PC) on DHA levels in the liver and brain of congenital n‐3‐deficient mice. RESULTS: Dietary supplementation with DHA significantly changed the fatty acid composition of various phospholipid molecules in the cerebral cortex and liver while DHA‐enriched phospholipid was more effective than DHA triglyceride (TG) in increasing brain and liver DHA. Both DHA‐PS and DHA‐PC could effectively increase the DHA levels, but DHA in the PS form was superior to PC in the contribution of DHA content in the brain ether‐linked PC (ePC) and liver lyso‐phosphatidylcholine molecular species. DHA‐PC showed more significant effects on the increase of DHA in liver TG, PC, ePC, phosphatidylethanolamine (PE) and PE plasmalogen (pPE) molecular species and decreasing the arachidonic acid level in liver PC plasmalogen, ePC, PE and pPE molecular species compared with DHA‐PS. CONCLUSION: The effect of dietary interventions with different molecular forms of DHA for brain and liver lipid profiles is different, which may provide theoretical guidance for dietary supplementation of DHA for people. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Bilateral communication between bones and muscles is essential for healing composite bone–muscle injuries from orthopedic surgeries and trauma. However, these injuries are often characterized by exaggerated inflammation, which can disrupt bone–muscle crosstalk, thereby seriously delaying the healing of either tissue. Existing approaches are largely effective at healing single tissues. However, simultaneous healing of multiple tissues remains challenging, with little research conducted to date. Here we introduce collagen patches that overcome this overlooked issue by harnessing the plasticity of macrophage phenotypes. Phosphatidylserine liposomes (PSLs) capable of shifting the macrophage phenotype from inflammatory M1 into anti-inflammatory/prohealing M2 were coated on collagen patches via a layer-by-layer method. Original collagen patches failed to improve tissue healing under inflammatory conditions coordinated by M1 macrophages. In contrast, PSL-coated collagen patches succeeded in accelerating bone and muscle healing by inducing a microenvironment dominated by M2 macrophages. In cell experiments, differentiation of preosteoblasts and myoblasts was completely inhibited by secretions of M1 macrophages but unaffected by those of M2 macrophages. RNA-seq analysis revealed that type I interferon and interleukin-6 signaling pathways were commonly upregulated in preosteoblasts and myoblasts upon stimulation with M1 macrophage secretions, thereby compromising their differentiation. This study demonstrates the benefit of PSL-mediated M1-to-M2 macrophage polarization for simultaneous bone and muscle healing, offering a potential strategy toward simultaneous regeneration of multiple tissues. Existing approaches for tissue regeneration, which primarily utilize growth factors, have been largely effective at healing single tissues. However, simultaneous healing of multiple tissues remains challenging and has been little studied. Here we demonstrate that collagen patches releasing phosphatidylserine liposomes (PSLs) promote M1-to-M2 macrophage polarization and are effective for simultaneous healing of bone and muscle. Transcriptome analysis using next-generation sequencing reveals that differentiation of preosteoblasts and myoblasts is inhibited by the secretions of M1 macrophages but promoted by those of M2 macrophages, highlighting the importance of timely regulation of M1-to-M2 polarization in tissue regeneration. These findings provide new insight to tissue healing of multiple tissues. [Display omitted] [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) and α-Linolenic acid (ALA), are positively associated with cognitive function, but their combination effects and possible mechanisms remain unclear. We aimed to explore the effects on cognition and potential mechanism of the supplements. This randomized, double-blind, placebo-controlled trial recruited 190 MCI patients in Tianjin, China, and randomly assigned in intervention group and placebo group. Each group consumed two capsules every day for 12 months. Each capsule for intervention group contains 144 mg ALA, 31.5 mg PS and 3.6 mg Ginkgo total flavonoids as main functional components, with 0.48 mg Vitamin B 1 (as thiamine hydrochloride), 0.48 mg Vitamin B 6 (as pyridoxine hydrochloride) and 90 μg folic acid as supplement. Capsules for placebo group were identical but contain no active ingredients. Cognitive function, serum n-3 polyunsaturated fatty acids (PUFAs) and neurotransmitters were assessed at baseline and 12 months. Linear mixed effects model and causal mediation analysis were conducted to explore the effects and potential mechanism of the intervention. A total of 190 participants (mean [SD] age, 67.95 [5.62] years; 70 (36.8 %) male and 120 (63.2 %) female) were randomized to the placebo group (n = 95) and intervention group (n = 95). Compared with placebo group, the intervention group had statistically significant improvements in arithmetic testing (β , 0.688; 95 % CI , 0.103–1.274), the similarity test (β , 1.070; 95 % CI , 0.472–1.667) and short-term memory (β , 0.600; 95 % CI , 0.399–0.800). Besides, the intervention group had statistically significant increases in serum ALA (β , 1.620; 95 % CI , 0.967–2.265), DHA (β , 2.797; 95 % CI , 1.075–4.532), EPA (β , 1.472; 95 % CI , 0.296–2.643), acetylcholine (β , 0.441; 95 % CI , 0.415–0.468), GABA (β , 0.009; 95 % CI , 0.001–0.016) and 5-HT (β , 0.160; 95 % CI , 0.081–0.238) compared to the placebo group. And the intervention may improve short-term memory by increasing serum ALA levels (average causal mediation effect = 0.132, 95 % CI , 0.053–0.225) with 19.7 % mediation proportion. This food supplement containing phosphatidylserine could improve different cognitive functions of MCI patients, especially short-term memory, and increase serum n-3 PUFAs and neurotransmitters levels. Serum ALA level might play a mediation role. • A food supplement containing phosphatidylserine could improve cognitive functions. • The supplement could increase serum n-3 PUFAs and neurotransmitters levels. • Serum ALA levels may mediate the improvement in short-term memory. [ABSTRACT FROM AUTHOR]

Abstract Macrophages are multifunctional innate immune cells that play indispensable roles in homeostasis, tissue repair, and immune regulation. However, dysregulated activation of macrophages is implicated in the pathogenesis of various human disorders, making them a potential target for treatment. Through the expression of pattern recognition and scavenger receptors, macrophages exhibit selective uptake of pathogens and apoptotic cells. Consequently, the utilization of drug carriers that mimic pathogenic or apoptotic signals shows potential for targeted delivery to macrophages. In this study, a series of mannosylated or/and phosphatidylserine (PS) -presenting liposomes were developed to target macrophages via the design of experiment (DoE) strategy and the trial-and-error (TaE) approach. The optimal molar ratio for the liposome formulation was DOPC: DSPS: Chol: PEG-PE = 20:60:20:2 based on the results of cellular uptake and cytotoxicity evaluation on RAW 264.7 and THP-1 in vitro. Results from in vivo distribution showed that, in the DSS-induced colitis model and collagen II-induced rheumatoid arthritis model, PS-presenting liposomes (PS-Lipo) showed the highest accumulation in intestine and paws respectively, which holds promising potential for macrophage target therapy since macrophages are abundant at inflammatory sites and contribute to the progression of corresponding diseases. Organs such as the heart, liver, spleen, lung, and kidney did not exhibit histological alterations such as inflammation or necrosis when exposed to PC-presenting liposomes (PC-Lipo) or PS-Lipo. In addition, liposomes demonstrated hemobiocompatibility and no toxicity to liver or kidney for circulation and did not induce metabolic injury in the animals. Thus, the well-designed PS-Lipo demonstrated the most potential for macrophage target therapy.

Microglia play a crucial role in synaptic elimination by engulfing dystrophic neurons via triggering receptors expressed on myeloid cells 2 (TREM2). They are also involved in the clearance of beta‐amyloid (Aβ) plaques in Alzheimer's disease (AD); nonetheless, the driving force behind TREM2‐mediated phagocytosis of beta‐amyloid (Aβ) plaques remains unknown. Here, using advanced 2D/3D/4D co‐culture systems with loss‐of‐function mutations in TREM2 (a frameshift mutation engineered in exon 2) brain organoids/microglia/assembloids, it is identified that the clearance of Aβ via TREM2 is accelerated by externalized phosphatidylserine (ePtdSer) generated from dystrophic neurons surrounding the Aβ plaques. Moreover, it is investigated whether microglia from both sporadic (CRISPR‐Cas9‐based APOE4 lines) and familial (APPNL‐G‐F/MAPT double knock‐in mice) AD models show reduced levels of TREM2 and lack of phagocytic activity toward ePtdSer‐positive Aβ plaques. Herein new insight is provided into TREM2‐dependent microglial phagocytosis of Aβ plaques in the context of the presence of ePtdSer during AD progression. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection. [ABSTRACT FROM AUTHOR]

Macrophages are multifunctional innate immune cells that play indispensable roles in homeostasis, tissue repair, and immune regulation. However, dysregulated activation of macrophages is implicated in the pathogenesis of various human disorders, making them a potential target for treatment. Through the expression of pattern recognition and scavenger receptors, macrophages exhibit selective uptake of pathogens and apoptotic cells. Consequently, the utilization of drug carriers that mimic pathogenic or apoptotic signals shows potential for targeted delivery to macrophages. In this study, a series of mannosylated or/and phosphatidylserine (PS) -presenting liposomes were developed to target macrophages via the design of experiment (DoE) strategy and the trial-and-error (TaE) approach. The optimal molar ratio for the liposome formulation was DOPC: DSPS: Chol: PEG-PE = 20:60:20:2 based on the results of cellular uptake and cytotoxicity evaluation on RAW 264.7 and THP-1 in vitro. Results from in vivo distribution showed that, in the DSS-induced colitis model and collagen II-induced rheumatoid arthritis model, PS-presenting liposomes (PS-Lipo) showed the highest accumulation in intestine and paws respectively, which holds promising potential for macrophage target therapy since macrophages are abundant at inflammatory sites and contribute to the progression of corresponding diseases. Organs such as the heart, liver, spleen, lung, and kidney did not exhibit histological alterations such as inflammation or necrosis when exposed to PC-presenting liposomes (PC-Lipo) or PS-Lipo. In addition, liposomes demonstrated hemobiocompatibility and no toxicity to liver or kidney for circulation and did not induce metabolic injury in the animals. Thus, the well-designed PS-Lipo demonstrated the most potential for macrophage target therapy. [ABSTRACT FROM AUTHOR]

Phosphatidylserine (PS) is an important anionic phospholipid that is synthesized within the endoplasmic reticulum (ER). While PS shows the highest enrichment and serves important functional roles in the plasma membrane (PM) but its role in the nucleus is poorly explored. Using three orthogonal approaches, we found that PS is also uniquely enriched in the inner nuclear membrane (INM) and the nuclear reticulum (NR). Nuclear PS is critical for supporting the translocation of CCTα and Lipin1α, two key enzymes important for phosphatidylcholine (PC) biosynthesis, from the nuclear matrix to the INM and NR in response to oleic acid treatment. We identified the PS-interacting regions within the M-domain of CCTα and M-Lip domain of Lipin1α, and show that lipid droplet formation is altered by manipulations of nuclear PS availability. Our studies reveal an unrecognized regulatory role of nuclear PS levels in the regulation of key PC synthesizing enzymes within the nucleus. Synopsis: Phosphatidylserine (PS) is a phospholipid mainly enriched in the inner leaflet of the plasma membrane; however, its role in the nucleus remains unclear. This study describes the presence of PS in the inner nuclear membrane and nuclear reticulum, where it promotes nuclear membrane association of two key enzymes of phosphatidylcholine (PC) biosynthesis in response to oleic acid treatment. PS biosensors show its enrichment in the inner nuclear membrane (INM) and the nuclear reticulum (NR). PS in the inner nuclear membrane is synthesized at the endoplasmic reticulum. Nuclear PS interacts with two PC biosynthetic enzymes, CCTα and Lipin1α, promoting their translocation to the inner nuclear membrane. Nuclear PS biases PC biosynthesis toward membrane expansion rather than lipid droplet formation. Phosphatidylserine supports inner nuclear membrane enrichment of phosphatidylcholine biosynthetic enzymes, CCTα and Lipin1α, for nuclear membrane expansion. [ABSTRACT FROM AUTHOR]

Background: Production of anti-phosphatidylserine (anti-PS) antibodies has been associated with malaria and can aggravate pathology. How these autoantibodies develop during early childhood in a malaria context is not known. We examined levels of anti-PS IgG and IgM antibodies in a longitudinal cohort of mother-baby pairs during birth, in the infants at 2.5, 6 months, and in mothers and their babies at 9 months postpartum. Results: There was no difference between levels of anti-PS IgG in cord blood and the mothers' peripheral blood at birth. However, anti-PS IgM levels were significantly higher in the mothers compared to the infants' cord blood, and IgM levels were steadily increasing during the first 9 months of the infants' life. In infants that had the highest anti-PS IgM levels at birth, there was a decline until 6 months with a rise at 9 months. Infants that possessed high anti-PS IgG at birth also exhibited a progressive decline in levels. When anti-PS were correlated to different fractions of B-cells, there were several correlations with P. falciparum specific atypical B cells both at birth and at 2.5 months for the infants, especially for anti-PS IgM. Anti-PS also correlated strongly to C1q-fixing antibodies at birth. Conclusion: These results show that anti-PS IgG acquired by mothers could be transferred transplacentally and that IgM antibodies targeting PS are acquired during the first year of life. These results have increased the knowledge about autoimmune responses associated with infections in early life and is critical for a comprehensive understanding of malaria vaccine functionality in endemic areas. [ABSTRACT FROM AUTHOR]

Despite progress in the management of patients with retinal vascular and degenerative diseases, there is still an unmet clinical need for safe and effective therapeutic options with novel mechanisms of action. Recent mechanistic insights into the pathogenesis of retinal diseases with a prominent vascular component, such as retinal vein occlusion (RVO), diabetic retinopathy (DR) and wet age-related macular degeneration (AMD), may open up new treatment paradigms that reach beyond the inhibition of vascular endothelial growth factor (VEGF). Phosphatidylserine (PS) is a novel lipid target that is linked to the pathophysiology of several human diseases, including retinal diseases. PS acts upstream of VEGF and complement signaling pathways. Annexin A5 is a protein that targets PS and inhibits PS signaling. This review explores the current understanding of the potential roles of PS as a target and Annexin A5 as a therapeutic. The clinical development status of Annexin A5 as a therapeutic and the potential utility of PS-Annexin A5 as a theranostic pairing in retinal vascular conditions in particular is described. [ABSTRACT FROM AUTHOR]

Alzheimer's disease is the fastest‐growing neurodegenerative disease that affects over six million Americans. The abnormal aggregation of amyloid β peptide and Tau protein is the expected molecular cause of the loss of neurons in brains of AD patients. A growing body of evidence indicates that lipids can alter the aggregation rate of amyloid β peptide and modify the toxicity of amyloid β aggregates. However, the role of lipids in Tau aggregation remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which phospatidylserine (PS) altered the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N terminal inserts that enhance the binding of Tau to tubulin. We found that the length and saturation of fatty acids (FAs) in PS altered the aggregation rate of 2N4R isoform, while no changes in the aggregation rate of 1N4R were observed. These results indicate that N terminal inserts play an important role in protein–lipid interactions. We also found that PS could change the toxicity of 1N4R and 2N4R Tau fibrils, as well as alter molecular mechanisms by which these aggregates exert cytotoxicity to neurons. Finally, we found that although Tau fibrils formed in the presence and absence of PS endocytosed by cells, only fibril species that were formed in the presence of PS exert strong impairment of the cell mitochondria. [ABSTRACT FROM AUTHOR]

Multidrug resistance significantly impedes the efficacy of cancer chemotherapy. Resistance often arises from the reduced cellular uptake of chemotherapeutic drugs, a process crucial for their cytotoxic effects. This reduction is frequently due to transmembrane efflux pumps powered by ATP from mitochondria and the cytoplasmic matrix, leading to lower intracellular concentrations of these drugs. This study introduces an amphiphilic molecule, bis(zinc-dipicolylamine) farnesol (Bis-ZnDPA), which targets phosphatidylserine (PS) – a negatively charged phospholipid prominently displayed on the outer leaflet of cancer cell plasma membranes. Integrating the hydrophobic segment of Bis-ZnDPA into the plasma membrane disrupts its integrity, potentially leading to hole formation and facilitating the uptake of chemotherapeutic drugs. Furthermore, the binding of Bis-ZnDPA to phosphatidylserine inhibits ATP production caused by Ca2+ influx and deregulation of the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway, reducing the efflux of drugs from cells. The results indicate the potent synergistic effect of Bis-ZnDPA with chemotherapeutic agents, suggesting that targeting PS is a viable strategy for overcoming multidrug resistance in cancer chemotherapy.

Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation, and the PM order are important factors for the stability of VP40 binding and oligomerization at the PM. The use of FDA-approved drugs to fluidize the PM destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Overall, these findings support an EBOV assembly mechanism that reaches beyond lipid headgroup specificity by using ordered PM lipid regions independent of cholesterol.

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.

Aim: Because of its involvement in membrane properties, annexin A5 has a significant impact on membrane-dependent processes within hemostasis, including the initiation of the coagulation cascade, the activation of factor X and prothrombin, and the function of protein C on membrane surfaces. By binding to phosphatidylserine, annexin A5 acts as a protective shield, masking exposed phospholipid surfaces and exerting a general anticoagulant effect. Considering the significant influence of annexin A5 on the activation of hemostatic compounds on cell surfaces, it is crucial to further investigate its mechanisms. Methods: After purification of polyHis-Tag proteins by immobilized metal affinity chromatography from E. coli Rosetta cell biomass, we checked the purity of the eluate by SDS-PAGE. We performed APTT test, PT test and platelet aggregation to find out the anticoagulation influence of recombinant annexin A5 in these tests. Results: Annexin in the highest concentration of 40 μg/ml prolonged blood plasma clotting time in activated partial thrombin time by almost 50%, blood plasma clotting time in prothrombin time by almost 15% and did not influence the rate of platelet aggregation. Conclusion: It can be assumed that its anticoagulant effect is directed to the enzyme complexes of the hemostasis system and does not extend to platelet receptors. The obtained protein can be used to study the activation of hemostatic components on cell surfaces.

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.

High-density lipoprotein (HDL) transports excess cholesterol from peripheral tissues back to the liver, and plasma HDL levels are inversely related to cardiovascular disease incidence. ATP-binding cassette A1 (ABCA1) is a member of the ABC protein superfamily, and generates nascent HDL, which consists of several hundreds of phospholipids and cholesterol wrapped by apolipoprotein A-I (apoA-I). However, it remains unclear whether cholesterol is a transport substrate of ABCA1. Since ATP hydrolysis of ABC proteins is typically increased by their transport substrates, we characterized the effects of cholesterol on the ATPase activity of purified ABCA1 using liposomes of various lipid compositions. ABCA1 showed substantial ATPase activity (20–30 nmol |$\cdot$| min−1 |$\cdot$| mg−1) only in liposomes containing anionic lipids, including phosphatidylserine. Cholesterol increased the ATPase activity by 1.6- to 3-fold in the presence of anionic lipids. Moreover, phosphatidylserine addition to BHK/ABCA1 cells increased phosphatidylcholine and cholesterol efflux to apoA-I. Next, we investigated the sterol specificity of ABCA1. The ATPase activity of ABCA1 was strongly enhanced by desmosterol and zymosterol, similar to cholesterol. In contrast, 7-dehydrocholesterol and lathosterol weakly increased the ATPase activity, and no increase was observed with stigmasterol or brassicasterol. These findings suggest that ABCA1 transports cholesterol and prefers cholesterol over plant sterols as a transport substrate. [ABSTRACT FROM AUTHOR]

Oxidized low‐density lipoprotein (oxLDL), a key component in atherosclerosis and hyperlipidemia, is a risk factor for atherothrombosis in dyslipidemia, yet its mechanism is poorly understood. In this study, we used oxLDL‐induced human aortic endothelial cells (HAECs) and high‐fat diet (HFD)‐fed mice as a hyperlipidemia model. Phosphatidylserine (PS) exposure, cytosolic Ca2+, reactive oxygen species (ROS), and lipid peroxidation were measured by flow cytometer. TMEM16F expression was detected by immunofluorescence, western blot, and reverse transcription polymerase chain reaction. Procoagulant activity (PCA) was measured by coagulation time, intrinsic/extrinsic factor Xase, and thrombin generation. We found that oxLDL‐induced PS exposure and the corresponding PCA of HAECs were increased significantly compared with control, which could be inhibited over 90% by lactadherin. Importantly, TMEM16F expression in oxLDL‐induced HAECs was upregulated by enhanced intracellular Ca2+ concentration, ROS, and lipid peroxidation, which led to PS exposure. Meanwhile, the knockdown of TMEM16F by short hairpin RNA significantly inhibited PS exposure in oxLDL‐induced HAECs. Moreover, we observed that HFD‐fed mice dramatically increased the progress of thrombus formation and accompanied upregulated TMEM16F expression by thromboelastography analysis, FeCl3‐induced carotid artery thrombosis model, and western blot. Collectively, these results demonstrate that TMEM16F‐mediated PS exposure may contribute to prothrombotic status under hyperlipidemic conditions, which may serve as a novel therapeutic target for the prevention of thrombosis in hyperlipidemia. Highlights: Oxidized low‐density lipoproteins (oxLDL) could increase phosphatidylserine (PS) exposure and procoagulant activity (PCA) of human aortic endothelial cells (HAECs) in a TMEM16F‐dependent manner.Increased intracellular Ca2+ and oxidative stress may contribute to TMEM16F‐mediated phospholipid scrambling.Hyperlipidemia exhibited a prothrombotic phenotype, which was likely related to TMEM16F‐mediated PS exposure on HAECs.Inhibition of TMEM16F or PS could be a novel therapeutic strategy to alleviate the thromboembolic complication of hyperlipidemia. [ABSTRACT FROM AUTHOR]

Scramblase Xk-related protein 8 (Xkr8) regulates the externalization of phosphatidylserine (PS) during apoptosis and holds a pivotal role in fostering tumor immunosuppression. Targeting Xkr8 in conjunction with chemotherapy demonstrated a novel avenue for amplifying antitumor immune response and overcoming chemo-immune resistance. Here we further evaluated this strategy by using a clinically relevant orthotopic model and elucidated the mechanism through in-depth single-cell RNA sequencing (scRNA-seq). We found that Xkr8 knockdown exhibited the potential to lead to immunogenic cell death (ICD) by impeding the normal clearance of apoptotic cells. Co-delivery of Xkr8 small interference RNA (siRNA) and a prodrug conjugate of 5-fluorouracil (5-Fu) and oxoplatin (FuOXP) showed remarkable therapeutic efficacy in an orthotopic pancreatic tumor model with increased infiltration of proliferative NK cells and activated macrophages in the tumor microenvironment (TME). Single-cell trajectory analysis further unveiled that tumor infiltrating CD8+ T cells are differentiated favorably to cytotoxic over exhausted phenotype after combination treatment. Our study sheds new light on the impact of Xkr8 knockdown on TME and solidifies the rationale of combining Xkr8 knockdown with chemotherapy to treat various types of cancers. [Display omitted] [ABSTRACT FROM AUTHOR]

This study was conducted to investigate the effects of dietary phosphatidylserine (PS) supplementation on the growth performance, stress response, non-specific immunity and antioxidant capacity of juvenile blunt snout bream (Megalobrama ambylcephala) cultured under a high stocking density. A 2 × 2 two-factorial design was adopted, including two stocking densities (10 and 20 fish/m3) and two dietary PS levels (0 and 50 mg/kg). After the 12-week feeding trial, the high stocking density significantly decreased the final weight; weight gain rate; specific growth rate; feed intake; nitrogen retention efficiency; plasma complement 3 (C3) level; albumin/globulin (ALB/GLB, A/G) ratio; activity of myeloperoxidase, lysozyme (LZM) and glutathione peroxidase (GPX); gpx transcription; and abundance of sirtuin3 (Sirt3) and nuclear factor erythroid-2-related factor 2 (Nrf2). However, it significantly increased the plasma levels of cortisol, glucose (GLU), lactic acid (LD), total protein and GLB; hepatic malondialdehyde (MDA) content; and sirt1 transcription. PS supplementation significantly increased the plasma ALB and C4 levels; the A/G ratio; the activity of LZM, CAT and GPX; the transcription of sirt1, nrf2, manganese-containing superoxide dismutase and catalase; and the Nrf2 abundance. However, it significantly decreased the plasma levels of cortisol, GLU and GLB, as well as the hepatic MDA content. In addition, there was a significant interaction between the stocking density and PS supplementation regarding the effects on the plasma LD, ALB, GLB and C3 levels; A/G ratio; hepatic CAT activity; and protein abundance of Sod2. In conclusion, PS supplementation can counteract the high stocking density-induced stress response, redox imbalance and immunosuppression in blunt snout bream. [ABSTRACT FROM AUTHOR]

DOX liposomes have better therapeutic effects and lower toxic side effects. The targeting ability of liposomes is one of the key factors affecting the therapeutic effect of DOX liposomes. This study developed two types of targeted liposomes. Sialic acid (SA)-modified liposomes were designed to target the highly expressed Siglec-1 receptor on tumor-associated macrophages surface. Phosphatidylserine (PS)-modified liposomes were designed to promote phagocytosis by monocyte-derived macrophages through PS apoptotic signaling. In order to assess and compare the therapeutic potential of different targeted pathways in the context of anti-tumor treatment, we compared four phosphatidylserine membrane materials (DOPS, DSPS, DPPS and DMPS) and found that liposomes prepared using DOPS as material could significantly improve the uptake ability of RAW264.7 cells for DOX liposomes. On this basis, normal DOX liposomes (CL-DOX) and SA-modified DOX liposomes (SAL-DOX), PS-modified DOX liposomes (PS-CL-DOX), SA and PS co-modified DOX liposomes (PS-SAL-DOX) were prepared. The anti-tumor cells function of each liposome on S180 and RAW264.7 in vitro was investigated, and it was found that SA on the surface of liposomes can increase the inhibitory effect. In vivo efficacy results exhibited that SAL-DOX and PS-CL-DOX were superior to other groups in terms of ability to inhibit tumor growth and tumor inhibition index, among which SAL-DOX had the best anti-tumor effect. Moreover, SAL-DOX group mice had high expression of IFN-γ as well as IL-12 factors, which could significantly inhibit mice tumor growth, improve the immune microenvironment of the tumor site, and have excellent targeted delivery potential. [ABSTRACT FROM AUTHOR]

Mounting evidence indicates that a physiological function of amyloid-β (Aβ) is to mediate neural activity-dependent homeostatic and competitive synaptic plasticity in the brain. I have previously summarized the lines of evidence supporting this hypothesis and highlighted the similarities between Aβ and anti-microbial peptides in mediating cell/synapse competition. In cell competition, anti-microbial peptides deploy a multitude of mechanisms to ensure both self-protection and competitor elimination. Here I review recent studies showing that similar mechanisms are at play in Aβ-mediated synapse competition and perturbations in these mechanisms underpin Alzheimer's disease (AD). Specifically, I discuss evidence that Aβ and ApoE, two crucial players in AD, co-operate in the regulation of synapse competition. Glial ApoE promotes self-protection by increasing the production of trophic monomeric Aβ and inhibiting its assembly into toxic oligomers. Conversely, Aβ oligomers, once assembled, promote the elimination of competitor synapses via direct toxic activity and amplification of "eat-me" signals promoting the elimination of weak synapses. I further summarize evidence that neuronal ApoE may be part of a gene regulatory network that normally promotes competitive plasticity, explaining the selective vulnerability of ApoE expressing neurons in AD brains. Lastly, I discuss evidence that sleep may be key to Aβ-orchestrated plasticity, in which sleep is not only induced by Aβ but is also required for Aβ-mediated plasticity, underlining the link between sleep and AD. Together, these results strongly argue that AD is a disease of competitive synaptic plasticity gone awry, a novel perspective that may promote AD research. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy. [ABSTRACT FROM AUTHOR]

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective drugs in the treatment of patients with chronic myeloid leukemia. However, based on clinical studies, ponatinib was associated with the development of thrombotic complications. Since endothelial cells (ECs) regulate blood coagulation, their abnormal phenotype may play a role in the development of thrombotic events. We here aimed to investigate the effect of ponatinib on the procoagulant activity of cultured endothelial cells in vitro. Human coronary artery endothelial cells (HCAECs) were incubated with 50, 150, and 1000 nM of ponatinib. Subsequently, phosphatidylserine (PS) exposure and endothelial microvesicles (EMVs) were measured by flow cytometry. In addition, EC- and EMV-dependent thrombin generation was analyzed. To investigate pro-apoptotic effects of ponatinib, the level of Bax and Bcl-xL proteins were studied using Western blot and F3, THBD, and VCAM1 mRNAs were quantified by qPCR. Therapeutic concentrations of ponatinib significantly increased PS expression on ECs and the amount of EMVs which significantly shortened the time parameters of thrombin generation. In addition, these changes were associated with an increased ratio of Bax and Bcl-xL proteins in the presence of the decreased THBD mRNA level. Overall, ponatinib enhances the procoagulant activity of ECs via inducing apoptosis, which may contribute to thrombotic events. [ABSTRACT FROM AUTHOR]

Background: Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less susceptible to resistance but are seldom considered for treatment of these diseases. Methods: Our overall objective was to systematically assess small molecules for host cell-targeting activity to restrict proliferation of intracellular parasites. We carried out a literature survey to identify small molecules annotated for host factors implicated in Plasmodium falciparum infection. Alongside P. falciparum, we implemented in vitro parasite susceptibility assays also in the zoonotic parasite Plasmodium knowlesi and the veterinary parasite Babesia divergens. We additionally carried out assays to test directly for action on RBCs apart from the parasites. To distinguish specific host-targeting antiparasitic activity from erythrotoxicity, we measured phosphatidylserine exposure and hemolysis stimulated by small molecules in uninfected RBCs. Results: We identified diverse RBC target-annotated inhibitors with Plasmodium-specific, Babesia-specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of Plasmodium spp. in RBCs. Some inhibitors exhibit antimalarial activity with transient exposure to RBCs prior to infection with parasites, providing evidence for host-targeting activity distinct from direct inhibition of the parasite. Conclusions: We report here characterization of small molecules for antiproliferative and host cell-targeting activity for malaria and babesiosis parasites. This resource is relevant for assessment of physiological RBC-parasite interactions and may inform drug development and repurposing efforts. [ABSTRACT FROM AUTHOR]

Immune response against malaria and the clearance of Plasmodium parasite relies on germinal-center-derived B cell responses that are temporally and histologically layered. Despite a well-orchestrated germinal center response, anti-Plasmodium immune response seldom offers sterilizing immunity. Recent studies report that certain pathophysiological features of malaria such as extensive hemolysis, hypoxia as well as the extrafollicular accumulation of short-lived plasmablasts may contribute to this suboptimal immune response. In this review, we summarize some of those studies and attempt to connect certain host intrinsic features in response to the malarial disease and the resultant gaps in the immune response. [ABSTRACT FROM AUTHOR]

Abstract Microglia play a crucial role in synaptic elimination by engulfing dystrophic neurons via triggering receptors expressed on myeloid cells 2 (TREM2). They are also involved in the clearance of beta‐amyloid (Aβ) plaques in Alzheimer's disease (AD); nonetheless, the driving force behind TREM2‐mediated phagocytosis of beta‐amyloid (Aβ) plaques remains unknown. Here, using advanced 2D/3D/4D co‐culture systems with loss‐of‐function mutations in TREM2 (a frameshift mutation engineered in exon 2) brain organoids/microglia/assembloids, it is identified that the clearance of Aβ via TREM2 is accelerated by externalized phosphatidylserine (ePtdSer) generated from dystrophic neurons surrounding the Aβ plaques. Moreover, it is investigated whether microglia from both sporadic (CRISPR‐Cas9‐based APOE4 lines) and familial (APPNL‐G‐F/MAPT double knock‐in mice) AD models show reduced levels of TREM2 and lack of phagocytic activity toward ePtdSer‐positive Aβ plaques. Herein new insight is provided into TREM2‐dependent microglial phagocytosis of Aβ plaques in the context of the presence of ePtdSer during AD progression.

Calmodulin (CaM) is a ubiquitous calcium-sensitive messenger in eukaryotic cells. It was previously shown that CaM possesses an affinity for diverse lipid moieties, including those found on CaM-binding proteins. These facts, together with our observation that CaM accumulates in membrane-rich protrusions of HeLa cells upon increased cytosolic calcium, motivated us to perform a systematic search for unmediated CaM interactions with model lipid membranes mimicking the cytosolic leaflet of plasma membranes. A range of experimental techniques and molecular dynamics simulations prove unambiguously that CaM interacts with lipid bilayers in the presence of calcium ions. The lipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) hold the key to CaM–membrane interactions. Calcium induces an essential conformational rearrangement of CaM, but calcium binding to the headgroup of PS also neutralizes the membrane negative surface charge. More intriguingly, PE plays a dual role—it not only forms hydrogen bonds with CaM, but also destabilizes the lipid bilayer increasing the exposure of hydrophobic acyl chains to the interacting proteins. Our findings suggest that upon increased intracellular calcium concentration, CaM and the cytosolic leaflet of cellular membranes can be functionally connected.

Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection.

BackgroundProduction of anti-phosphatidylserine (anti-PS) antibodies has been associated with malaria and can aggravate pathology. How these autoantibodies develop during early childhood in a malaria context is not known. We examined levels of anti-PS IgG and IgM antibodies in a longitudinal cohort of mother-baby pairs during birth, in the infants at 2.5, 6 months, and in mothers and their babies at 9 months postpartum.ResultsThere was no difference between levels of anti-PS IgG in cord blood and the mothers’ peripheral blood at birth. However, anti-PS IgM levels were significantly higher in the mothers compared to the infants’ cord blood, and IgM levels were steadily increasing during the first 9 months of the infants’ life. In infants that had the highest anti-PS IgM levels at birth, there was a decline until 6 months with a rise at 9 months. Infants that possessed high anti-PS IgG at birth also exhibited a progressive decline in levels. When anti-PS were correlated to different fractions of B-cells, there were several correlations with P. falciparum specific atypical B cells both at birth and at 2.5 months for the infants, especially for anti-PS IgM. Anti-PS also correlated strongly to C1q-fixing antibodies at birth.ConclusionThese results show that anti-PS IgG acquired by mothers could be transferred transplacentally and that IgM antibodies targeting PS are acquired during the first year of life. These results have increased the knowledge about autoimmune responses associated with infections in early life and is critical for a comprehensive understanding of malaria vaccine functionality in endemic areas.

Over the past 40 years, research has heavily emphasized stroke treatments that directly target ischemic cascades after stroke onset. Much attention has focused on studying neuroprotective drugs targeting one aspect of the ischemic cascade. However, the single-target therapeutic approach resulted in minimal clinical benefit and poor outcomes in patients. Considering the ischemic cascade is a multifaceted and complex pathophysiological process with many interrelated pathways, the spotlight is now shifting towards the development of neuroprotective drugs that affect multiple aspects of the ischemic cascade. Phosphatidylserine (PS), known as the “eat-me” signal, is a promising candidate. PS is involved in many pathophysiological changes in the central nervous system after stroke onset, including apoptosis, inflammation, coagulation, and neuronal regeneration. Moreover, PS might also exert various roles in different phases after stroke onset. In this review, we describe the synthesis, regulation, and function of PS under physiological conditions. Furthermore, we also summarize the different roles of PS after stroke onset. More importantly, we also discuss several treatment strategies that target PS. We aim to advocate a novel stroke care strategy by targeting PS through a translational perspective.

Aim. Considering the significant influence of annexin A5 on the activation of hemostatic compounds on cell surfaces, it becomes crucial to delve deeper into its mechanisms. Therefore, the objective of our study was twofold: first, to produce recombinant annexin A5, and second, to explore its impact on platelet aggregation and blood plasma clotting. Through this investigation, we aim to shed light on the intricate interplay between annexin A5 and the hemostatic system, potentially uncovering novel insights into thrombosis and hemostasis regulation. Materials and Methods. Accumulation of cell biomass for the production of recombinant annexin А5. E. coli strain Rosetta (DE3) pET28a-EGFP-Annexin A5 was poured into 50 mL tube with 20 ml of lysogeny broth (LB) medium and the kanamycin were added to a concentration of 50 μg/ml of medium. The culture was allowed to grow overnight under active aeration and at 37 °C. Then 5 ml of the overnight culture was taken and 200 mL fresh LB medium was mixed and kanamycin to a final concentration of 50 μg/ml of medium was added. It was cultured at 37 °C under active aeration to an optical density (OD) of 0.3–0.5 at 600 nm. After reaching this optical density, temperature was lowered to 30 °C, an inducer of recombinant protein expression IPTG (Isopropyl β- d-1- thiogalactopyranoside) with a final concentration of 1 mM was added and culture flasks were rotated for 3 hours with active aeration. After that, the medium was centrifuged for 20 minutes at 3600 rpm. The supernatant was decanted, and the cells’ precipitate was stored at –80 °C. A green precipitate was obtained, which at first glance indicates a successful protein expression. Purification of polyHis-Tag proteins by immobilized metal affinity chromatography. The column with nickel-agarose was deconditioned by successive washing with 5-10 column volumes of ethanol, distilled water, 0.5 NaOH, and distilled water. Then it was equilibrated with 15–20 volumes of wash buffer (50 mM Na2HPO4, 0.3 M NaCl, 10 mM imidazole, pH 8.0) with 6 M urea. The precipitate of producer bacteria from 50 ml of bacterial suspension was dissolved in 1 ml of wash buffer. The mixture was sonicated 3 times for 20 seconds each using an ultrasonic disintegrator, and the microtubes were kept on ice. The mixture was centrifuged for 20 min at 13400 rpm. The supernatant was applied to a nickel-agarose column. It was washed with 5–10 volumes of wash buffer (50 mM Na2HPO4, 0.3 M NaCl, 10 mM imidazole, 6 M urea), after that concentration of urea was gradually reduced with 1 M difference among solutions). Protein was eluted with wash buffer (50 mM Na2HPO4, 0.3 M NaCl, 250 mM imidazole). Results and Discussion. Analysis of recombinant annexin А5 production and purification. To analyze the purity of annexin A5 at different stages of purification, we tested lysate, eluted samples, and dialysis samples. In Fig. 1 we can observe presence of annexin A5 in the E. coli lysate. Affinity chromatography significantly purified the protein from impurities, but not completely. The effect of annexin on blood clotting time. As shown in Fig. 2, recombinant annexin A5 prolonged time of blood clotting induced by APTT-reagent. Annexin A5 also slightly prolonged blood clotting time induced be thromboplastin in PT test but not significantly. Conclusions. Our EGFP-conjugated annexin A5 prolonged blood clotting time in the APTT test, so it had the biological effect of natural annexin, but did not inhibit platelet activation and aggregation. It can be assumed that its anticoagulant effect is directed to the enzyme complexes of the hemostasis system and does not extend to platelet receptors. The obtained protein can be used to study the activation of hemostatic components on cell surfaces. [ABSTRACT FROM AUTHOR]

The search for protective substances that can be used during red blood cell thawing and the study of their effects on red blood cells contribute to increasing the number and quality of viable cells after the cryopreservation cycle. We studied the effect of posthypertonic shock and amphiphilic compounds on the shape and eryptosis of human erythrocytes. The method of flow cytometry was used, this allows analyzing two parameters simultaneously, which increases the efficiency of research. The shape was assessed by the sphericity index (SphI), and eryptosis by the redistribution of phosphatidylserine to the membrane outer surface. It has been shown that sodium decylsulfate and chlorpromazine reduce erythrocyte damage in posthypertonic shock by 3.6 and 4.2 times, respectively. Sodium decylsulfate helps to preserve the shape of cells (SphI coefficient remains the same), while when chlorpromazine is used, the shape changes towards spherical (SphI coefficient changes 2 times). The study of the level of Annexin V FITC binding to phosphatidylserine in outer layer of membrane revealed a concentration-dependent increase in fluorescence when sodium decylsulfate was used, indicating a disorder of the bilayer asymmetry. In contrast, chlorpromazine did not change the distribution of phosphatidylserine. Comparison of two parameters of cell viability - the sphericity coefficient and anexin binding - allowed us to choose the conditions that are optimal for the use of the studied protective substances. Namely, it is advisable to use the lowest effective concentration of sodium decylsulfate (200 mcmol/l) for protective purposes. This ensures the preservation of the cell shape and minimal impact on the membrane asymmetry [ABSTRACT FROM AUTHOR]

The receptor tyrosine kinase Tyro3 is abundantly expressed in neurons of the neocortex, hippocampus, and striatum, but its role in these cells is unknown. We found that neuronal expression of this receptor was markedly up-regulated in the postnatal mouse neocortex immediately prior to the final development of glutamatergic synapses. In the absence of Tyro3, cortical and hippocampal synapses never completed end-stage differentiation and remained electrophysiologically and ultrastructurally immature. Tyro3-/- cortical neurons also exhibited diminished plasma membrane expression of the GluA2 subunits of AMPA-type glutamate receptors, which are essential to mature synaptic function. Correspondingly, GluA2 membrane insertion in wild-type neurons was stimulated by Gas6, a Tyro3 ligand widely expressed in the postnatal brain. Behaviorally, Tyro3-/- mice displayed learning enhancements in spatial recognition and fear-conditioning assays. Together, these results demonstrate that Tyro3 promotes the functional maturation of glutamatergic synapses by driving plasma membrane translocation of GluA2 AMPA receptor subunits. [ABSTRACT FROM AUTHOR]

An environmentally friendly preparation technique was used to explore the mechanism of action of hybrid hydrophilic bimetallic gold-platinum (Au@Pt-NPs) against human cancer cell lines, followed by conjugation with doxorubicin (DOX). The synthesized DOX-loaded Au@Pt-NPs (DOX/Au@Pt-NPs) were examined using transmission electron microscopy, X-ray diffraction, and UV–vis spectroscopy to determine their physicochemical characteristics. Using a straightforward incubation method, phytochemicals-mediated capping efficiency and loading of DOX onto the Au@Pt-NPs surface (84%) was demonstrated by FT-IR spectroscopy. Studies on the release kinetics of DOX under acidic tumor conditions revealed a pH-regulated dependence response. According to the in vitro anticancer assessment, human cancer cells treated with DOX-loaded Au@Pt-NPs demonstrated a 3-fold increase in apoptosis compared to DOX treatment alone. Additionally, the DOX/Au@Pt-NPs nanocarrier demonstrated a time-dependent increase in phosphatidylserine flipping, an apoptosis induction marker, and a cell-specific reaction in HeLa and HepG2 cells, according to luminescent results from a real-time, bioluminescent recombinant annexin V test. According to the results, the manufacturing process is simple, and the hydrophilic Au@Pt-NPs were successful in DOX delivery and loading against human cancer cell lines, indicating their prospective as a supplementary for cervical and hepatocellular cancer therapy. [Display omitted] • We fabricated bimetallic gold-platinum (Au@Pt-NPs) conjugation with doxorubicin (DOX). • Synthesized DOX-loaded Au@Pt-NPs (DOX/Au@Pt-NPs) were examined using various spectral methods. • Studies on the release kinetics of DOX under acidic tumor conditions revealed a pH-regulated response. • Human cancer cells treated with DOX-loaded Au@Pt-NPs saw a 3-fold increase in apoptosis. • DOX/Au@Pt-NPs nanocarrier demonstrated a time-released phosphatidylserine exposure, an apoptosis induction. [ABSTRACT FROM AUTHOR]

Nowadays phospholipids are widely used as hepatoprotective, neuroprotective and anti-stress drugs, as well as the dietary supplements. Besides, lecithin consisting up to 70% of the phospholipids mixture: phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and phosphatidic acid, is the often component of food production as an emulsifier. Dose of these biologically active substances in the modern human diet could be quite high. Previously we have shown that chronic intestinal inflammation in Muc2-knockout mice induces behavioral changes along with the significant increase in the content of phospholipids in intestinal epithelial cells, particularly, phosphatidylcholine, phosphatidylserine and phosphatidic acid. Here we investigate the effects of long-term administration of a mixture of these phospholipids, as well as the effects of long-term administration of soy lecithin on the behavioral patterns in laboratory mice. Animals long-term taken a phospholipid mixture shows no normally observed preference towards females in the two intruders test (with female and male). In the social odor preference test, they also did not distinguish female and male odors, while non-social odors discrimination preserved. In addition, we identified a decrease in anxiety, obsessive traits, and schizophrenia-like behavior traits in these animals. Soy lecithin supplementation had similar effects on social behavior and compulsive traits, and increased aggression in males. Thus, long-term perinatal administration of either mixture of phospholipids (phosphatidylcholine, phosphatidylserine and phosphatidic acid) or soy lecithin can influence various aspects of behavior in mice. [ABSTRACT FROM AUTHOR]

Platelet lipid rafts are critical membrane domains for adhesion, aggregation, and clot retraction. Lipid rafts are isolated as a detergent-resistant membrane fraction via sucrose density gradient centrifugation. The platelet detergent-resistant membrane shifted to a higher density on the sucrose density gradient upon thrombin stimulation. The shift peaked at 1 min and returned to the control level at 60 min. During this time, platelets underwent clot retraction and spreading on a fibronectin-coated glass strip. Thrombin induced the transient tyrosine phosphorylation of several proteins in the detergent-resistant membrane raft fraction and the transient translocation of fibrin and myosin to the detergent-resistant membrane raft fraction. The level of phosphatidylserine (36:1) was increased and the level of phosphatidylserine (38:4) was decreased in the detergent-resistant membrane raft fraction via the thrombin stimulation. Furthermore, Glanzmann's thrombasthenia integrin αIIbβ3-deficient platelets underwent no detergent-resistant membrane shift to a higher density upon thrombin stimulation. As the phosphorylation of the myosin regulatory light chain on Ser19 was at a high level in Glanzmann's thrombasthenia resting platelets, thrombin caused no further phosphorylation of the myosin regulatory light chain on Ser19 or clot retraction. These observations suggest that the fibrin–integrin αIIbβ3–myosin axis and compositional change of phosphatidylserine species may be required for the platelet detergent-resistant membrane shift to a higher density upon stimulation with thrombin. [ABSTRACT FROM AUTHOR]

Washed red blood cells (RBCs) can be used to treat immune-related diseases. However, whether the washing process changes the quality of RBCs and affects the curative effect of transfusion therapy remains unclear. We retrospectively analysed the clinical data of patients who received blood transfusion. The physiological and biochemical parameters of RBCs were tested on an automated haematology-biochemical analyser. CD47 and phosphatidylserine (PS) plasma membrane expression were analysed using flow cytometry. Morphological changes in RBCs were observed using scanning electron microscopy. The results showed that the curative effect on patients who received washed RBCs was weaker than that on those who received non-washed RBCs. Physiological and biochemical parameters of RBCs were not significantly different. RBC immune indices changed significantly after washing. The expression of “don't eat me” signals was weakened, whereas the intensity of “eat me” signals was enhanced. This study suggests that the current use of physiological and biochemical parameters as indicators to evaluate the quality of RBCs may not be comprehensive and that evaluation of the real status of RBCs requires other effective parameters. Immune molecules in RBCs are expected to become supplementary markers for evaluating RBC quality.

Glycerophospholipids have emerged as a significant contributor to the intracellular growth of pathogenic protist Toxoplasma gondii. Phosphatidylserine (PtdSer) is one such lipid, attributed to the locomotion and motility-dependent invasion and egress events in its acutely infectious tachyzoite stage. However, the de novo synthesis of PtdSer and the importance of the pathway in tachyzoites remain poorly understood. We show that a base-exchange-type PtdSer synthase (PSS) located in the parasite’s endoplasmic reticulum produces PtdSer, which is rapidly converted to phosphatidylethanolamine (PtdEtn) by PtdSer decarboxylase (PSD) activity. The PSS-PSD pathway enables the synthesis of several lipid species, including PtdSer (16:0/18:1) and PtdEtn (18:2/20:4, 18:1/18:2 and 18:2/22:5). The PSS-depleted strain exhibited a lower abundance of the major ester-linked PtdEtn species and concurrent accrual of host-derived ether-PtdEtn species. Most phosphatidylthreonine (PtdThr) species—an exclusive natural analog of PtdSer, also made in the endoplasmic reticulum—were repressed. PtdSer species, however, remained largely unaltered, likely due to the serine-exchange reaction of PtdThr synthase in favor of PtdSer upon PSS depletion. Not least, the loss of PSS abrogated the lytic cycle of tachyzoites, impairing the cell division, motility, and egress. In a nutshell, our data demonstrate a critical role of PSS in the biogenesis of PtdSer and PtdEtn species and its physiologically essential repurposing for the asexual reproduction of a clinically relevant intracellular pathogen.

ABSTRACTSystemic infections by Candida spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from Candida albicans is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an in vitro nucleotidase-coupled malachite-green-based high throughput assay for purified C. albicans Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average Z’ score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against C. albicans cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting in vivo Cho1 function by inducing phenotypes consistent with the cho1∆∆ mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a Ki of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.IMPORTANCEFungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a Ki value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.