Glycerophospholipid remodeling is critical for orthoflavivirus infection.

Flavivirus infection is tightly connected to host lipid metabolism. Here, we performed shotgun lipidomics of cells infected with neurotropic Zika, West Nile, and tick-borne encephalitis virus, as well as dengue and yellow fever virus. Early in infection specific lipids accumulate, e.g., neutral lipids in Zika and some lysophospholipids in all infections. Ceramide levels increase following infection with viruses that cause a cytopathic effect. In addition, fatty acid desaturation as well as glycerophospholipid metabolism are significantly altered. Importantly, depletion of enzymes involved in phosphatidylserine metabolism as well as phosphatidylinositol biosynthesis reduce orthoflavivirus titers and cytopathic effects while inhibition of fatty acid monounsaturation only rescues from virus-induced cell death. Interestingly, interfering with ceramide synthesis has opposing effects on virus replication and cytotoxicity depending on the targeted enzyme. Thus, lipid remodeling by orthoflaviviruses includes distinct changes but also common patterns shared by several viruses that are needed for efficient infection and replication.

Phosphatidylserine synthase plays a critical role in the utilization of n-alkanes in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica can assimilate n-alkane as a carbon and energy source. To elucidate the significance of phosphatidylserine (PS) in the utilization of n-alkane in Y. lipolytica, we investigated the role of the Y. lipolytica ortholog (PSS1) of Saccharomyces cerevisiae PSS1/CHO1, which encodes a PS synthase. The PSS1 deletion mutant (pss1Δ) of Y. lipolytica could not grow on minimal medium in the absence of ethanolamine and choline but grew when either ethanolamine or choline was supplied to synthesize phosphatidylethanolamine and phosphatidylcholine. The pss1Δ strain exhibited severe growth defects on media containing n-alkanes even in the presence of ethanolamine and choline. In the pss1Δ strain, the transcription of ALK1, which encodes a primary cytochrome P450 that catalyses the hydroxylation of n-alkanes in the endoplasmic reticulum, was upregulated by n-alkane as in the wild-type strain. However, the production of functional P450 was not detected, as indicated by the absence of reduced CO-difference spectra in the pss1Δ strain. PS was undetectable in the lipid extracts of the pss1Δ strain. These results underscore the critical role of PSS1 in the biosynthesis of PS, which is essential for the production of functional P450 enzymes involved in n-alkane hydroxylation in Y. lipolytica.

HIV-1 Gag Polyprotein Affinity to the Lipid Membrane Is Independent of Its Surface Charge.

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

Mixtures of Intrinsically Disordered Neuronal Protein Tau and Anionic Liposomes Reveal Distinct Anionic Liposome-Tau Complexes Coexisting with Tau Liquid-Liquid Phase-Separated Coacervates.

Tau, an intrinsically disordered neuronal protein and polyampholyte with an overall positive charge, is a microtubule (MT) associated protein that binds to anionic domains of MTs and suppresses their dynamic instability. Aberrant tau-MT interactions are implicated in Alzheimer's and other neurodegenerative diseases. Here, we studied the interactions between full-length human protein tau and other negatively charged binding substrates, as revealed by differential interference contrast (DIC) and fluorescence microscopy. As a binding substrate, we chose anionic liposomes (ALs) containing either 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS, -1e) or 1,2-dioleoyl-sn-glycero-3-phosphatidylglycerol (DOPG, -1e) mixed with zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) to mimic anionic plasma membranes of axons where tau resides. At low salt concentrations (0 to 10 mM KCl or NaCl) with minimal charge screening, reaction mixtures of tau and ALs resulted in the formation of distinct states of AL-tau complexes coexisting with liquid-liquid phase-separated tau self-coacervates arising from the polyampholytic nature of tau containing cationic and anionic domains. AL-tau complexes (i.e. tau-lipoplexes) exhibited distinct types of morphologies. This included large ∼20-30 µm tau-decorated giant vesicles with additional smaller liposomes with bound tau attached to the giant vesicles and tau-mediated finite-size assemblies of small liposomes. As the salt concentration was increased to near and above 150 mM for 1:1 electrolytes, AL-tau complexes remained stable, while tau self-coacervate droplets were found to dissolve, indicative of the breaking of (anionic/cationic) electrostatic bonds between tau chains due to increased charge screening. The findings are consistent with the hypothesis that distinct cationic domains of tau may interact with anionic lipid domains of the lumen-facing monolayer of the axon's plasma membrane, suggesting the possibility of transient yet robust interactions near relevant ionic strengths found in neurons.

Efficacy of DHA-enriched phosphatidylserine and its underlying mechanism in alleviating polystyrene nanoplastics-induced hepatotoxicity in mice.

OBJECTIVE: Plastic pollution has become a global pollution problem that cannot be ignored. As the main destination of human oral intake, the toxic effects of plastic on the digestive system represented by the intestine and liver are the focus of current research. Marine-derived DHA-PS has a variety of biological activities, mainly focusing on improving brain function and regulating lipid metabolism. However, whether it has an improvement effect on PS-NPs-induced hepato-intestinal injury and the underlying mechanism remain unclear. METHODS: A murine liver injury model was established by gavage of PS-NPs for six weeks. By integrating approaches from lipidomics, transcriptomics, and gut microbiota analysis, the molecular mechanism by which DHA-PS alleviates PS-NPs-induced murine hepatotoxicity was explored through the "gut-liver axis". RESULTS: Our findings reveal that prolonged exposure to PS-NPs results in significant murine liver damage and dysfunction, characterized by increased oxidative stress and inflammation, along with exacerbated hepatic lipid accumulation. Mechanistically, PS-NPs disrupt the hepatic SIRT1-AMPK pathway by suppressing the expression of SIRT1, AMPKα, and PPARα, while enhancing the expression of SREBP-1c, ultimately leading to disordered hepatic lipid metabolism. The sphingolipid and glycerophospholipid metabolic pathways were particularly affected. Additionally, in agreement with transcriptomic analyses, PS-NPs activate the hepatic TLR4/NF-κB pathway. At the same time, exposure to PS-NPs decreases the expression of ZO-1, occludin, and claudin-1, diminishes the relative abundance of beneficial gut bacteria (norank_f_Muribaculaceae, Akkermansia, and norank_f_norank_o_Clostridia_UCG-014), and increases the prevalence of pathogenic gut bacteria (Coriobacteriaceae_UCG-002 and Desulfovibrio), exacerbating liver injury through the gut-liver axis. However, administering DHA-PS (50 mg/kg) effectively alleviated these injuries. CONCLUSION: This study was the first to employ multi-omics techniques to elucidate the potential mechanisms underlying hepatotoxicity induced by PS-NPs, thereby supporting the use of DHA-PS as a dietary supplement to mitigate the effects of nanoplastic pollutants.

Can calmodulin bind to lipids of the cytosolic leaflet of plasma membranes?

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.

Phosphatidylserine liposomes induce a phagosome acidification-dependent and ROS-mediated intracellular killing of Mycobacterium abscessus in human macrophages.

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.

SARS-CoV-2 spike fusion peptide trans interaction with phosphatidylserine lipid triggers membrane fusion for viral entry.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the fusion machine for host cell entry. Still, the mechanism by which spike protein interacts with the target lipid membrane to facilitate membrane fusion during entry is not fully understood. Here, using steady-state membrane fusion and single-molecule fluorescence resonance energy transfer imaging of spike trimers on the surface of SARS-CoV-2 pseudovirion, we directly show that spike protein interacts with phosphatidylserine (PS) lipid in the target membrane for mediating fusion. We observed that the fusion peptide of the spike S2 domain interacts with the PS lipid of the target membrane. Low pH and Ca2+ trigger the spike conformational change and bring fusion peptide in close proximity to the PS lipid of the membrane. The binding of the spike with PS lipid of its viral membrane (cis interaction) impedes the fusion activation. PS on the target membrane promotes spike binding via trans interaction, prevents the cis interaction, and accelerates fusion. Sequestering or absence of PS lipid abrogates the spike-mediated fusion process and restricts SARS-CoV-2 infectivity. We found that PS-dependent interaction for fusion is conserved across all the SARS-CoV-2 spike variants of concern (D614G, Alpha, Beta, Delta, and Omicron). Our study suggests that PS lipid is indispensable for SARS-CoV-2 spike-mediated virus and target membrane fusion for entry, and restricting PS interaction with spike inhibits the SARS-CoV-2 spike-mediated entry. Therefore, PS is an important cofactor and acts as a molecular beacon in the target membrane for SARS-CoV-2 entry. IMPORTANCE: The role of lipids in the host cell target membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is not clear. We do not know whether SARS-CoV-2 spike protein has any specificity in terms of lipid for membrane fusion reaction. Here, using in vitro reconstitution of membrane fusion assay and single-molecule fluorescence resonance energy transfer imaging of SARS-CoV-2 spike trimers on the surface of the virion, we have demonstrated that phosphatidylserine (PS) lipid plays a key role in SARS-CoV-2 spike-mediated membrane fusion reaction for entry. Membrane-externalized PS lipid strongly promotes spike-mediated membrane fusion and COVID-19 infection. Blocking externalized PS lipid with PS-binding protein or in the absence of PS, SARS-CoV-2 spike-mediated fusion is strongly inhibited. Therefore, PS is an important target for restricting viral entry and intervening spike, and PS interaction presents new targets for COVID-19 interventions.

Red Blood Cell Membrane Spontaneously Coated Nanoprodrug Based on Phosphatidylserine for Antiatherosclerosis Applications.

Atherosclerosis (AS) is characterized by the accumulation of lipids within the walls of coronary arteries, leading to arterial narrowing and hardening. It serves as the primary etiology and pathological basis for cardiovascular diseases affecting the heart and brain. However, conventional pharmacotherapy is constrained by inadequate drug delivery and pronounced toxic side effects. Moreover, the inefficacy of nanomedicine delivery systems in controlling disease progression may be attributed to nonspecific clearance by the mononuclear phagocyte system. Thus, a biomimetic platform spontaneously enveloped by red blood cell membrane is exploited for anti-atherosclerosis applications, offering favorable biocompatibility. The CLIKKPF polypeptide is introduced to develop red blood cell membrane spontaneously encapsulated nanotherapeutics only through simple coincubation. Given the functional modifications, RBC@P-LVTNPs is beneficial to facilitate the target drug delivery to the atherosclerotic lesion, responding precisely to the pathological ROS accumulation, thereby accelerating the on-demand drug release. Both in vivo and in vitro results also confirm the significant therapeutic efficacy and favorable biocompatibility of the biomimetic nanomedicine delivery system, thus providing a promising candidate for nanotherapeutics against AS.

Acquisition of anti-phosphatidylserine IgM and IgG antibodies by infants and their mothers over time in Uganda.

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.

Phosphatidylserine-functional polydimethylsiloxane substrates regulate macrophage M2 polarization via modulus-dependent NF-κB/PPARγ pathway.

Macrophages, highly plastic innate immune cells, critically influence the success of implantable devices by responding to biochemical and physical cues. However, the mechanisms underlying their synergistic regulation of macrophage polarization on implant surfaces remain poorly understood. Therefore, we constructed anti-inflammatory phosphatidylserine (PS) modified polydimethylsiloxane (PDMS) substrates with low, medium, and high modulus (1-100 kPa) to investigate the combined effects and underlying mechanisms of substrate modulus and biochemical signal on macrophage polarization. The introduction of PS on the PDMS surface not only significantly enhanced the polarization of M0 to M2 but also potently suppressed lipopolysaccharide (LPS)-induced M1 activation, with this effect further potentiated by a reduction in substrate modulus. In vivo subcutaneous implantation experiments also corroborated the synergistic effect of PS functionalization and low modulus PDMS in inhibiting M1 activation and promoting M2 polarization. Notably, reduced modulus led to decreased integrin αV/ß3 clustering and cytoskeletal protein aggregation, ultimately diminishing YAP activation and nuclear translocation. Concomitantly, this disruption of the Piezo1-cytoskeletal protein positive feedback loop resulted in reduced p65/IκB phosphorylation and inflammation, while concurrently promoting PPARγ expression. Overall, our findings underscore the pivotal role of substrate modulus in modulating PS-mediated biomaterial-cell interactions, synergistically potentiating PS-induced M2 macrophage polarization, thus paving the way for the design of advanced immunomodulatory biomaterials.

Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

BACKGROUND: Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. METHODS: Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. RESULTS: External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. CONCLUSIONS: The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

The non-enzymatic oxidation of phosphatidylethanolamine and phosphatidylserine and their intriguing roles in inflammation dynamics and diseases.

Phosphatidylethanolamine (PE) and phosphatidylserine (PS), along with phosphatidylcholine (PC), are key phospholipids (PL) in cell membranes and lipoproteins, prone to oxidative modifications. Their oxidized forms, OxPE and OxPS, play significant roles in inflammation and immune response. This review explores their structural oxidative changes under non-enzymatic conditions and their roles in physiological and pathological contexts, influencing inflammation, and immunity. Specific oxidations of PE and PS significantly alter their physicochemical properties, leading to enhanced biological functions, reduced activity, or inactivation. OxPE may show pro-inflammatory actions, similar to well-documented OxPC, while the OxPS pro-inflammatory effects are less noted. However, OxPS and OxPE have also shown an antagonistic effect against lipopolysaccharides (LPS), suggesting a protective role against exacerbated immune responses, similar to OxPC. Further research is needed to deepen our understanding of these less-studied OxPL classes. The role of OxPE and OxPS in disease pathogenesis remains largely unexplored, with limited studies linking them to Alzheimer's disease, diabetes, rheumatoid arthritis, traumatic brain injury, and skin inflammation. These findings highlight the potential of OxPE and OxPS as biomarkers for disease diagnosis, monitoring, and therapeutic targeting.

Phosphatidylserine: A comprehensive overview of synthesis, metabolism, and nutrition.

Phosphatidylserine (PtdS) is classified as a glycerophospholipid and a primary anionic phospholipid and is particularly abundant in the inner leaflet of the plasma membrane in neural tissues. It is synthesized from phosphatidylcholine or phosphatidylethanolamine by exchanging the base head group with serine, and this reaction is catalyzed by PtdS synthase-1 and PtdS synthase-2 located in the endoplasmic reticulum. PtdS exposure on the outside surface of the cell is essential for eliminating apoptotic cells and initiating the blood clotting cascade. It is also a precursor of phosphatidylethanolamine, produced by PtdS decarboxylase in bacteria, yeast, and mammalian cells. Furthermore, PtdS acts as a cofactor for several necessary enzymes that participate in signaling pathways. Beyond these functions, several studies indicate that PtdS plays a role in various cerebral functions, including activating membrane signaling pathways, neuroinflammation, neurotransmission, and synaptic refinement associated with the central nervous system (CNS). This review discusses the occurrence of PtdS in nature and biosynthesis via enzymes and genes in plants, yeast, prokaryotes, mammalian cells, and the brain, and enzymatic synthesis through phospholipase D (PLD). Furthermore, we discuss metabolism, its role in the CNS, the fortification of foods, and supplementation for improving some memory functions, the results of which remain unclear. PtdS can be a potentially beneficial addition to foods for kids, seniors, athletes, and others, especially with the rising consumer trend favoring functional foods over conventional pills and capsules. Clinical studies have shown that PtdS is safe and well tolerated by patients.

Development of betabodies: The next generation of phosphatidylserine targeting agents.

Externalized phosphatidylserine (PS) is a phospholipid and a selective marker of the tumor microenvironment (TME). It is exposed on the outer leaflet of the plasma membrane of tumor-associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions in part to suppress immune responses by binding to PS receptors expressed on tumor-infiltrating myeloid cells. PS has been targeted with antibodies, such as bavituximab, that bind the phospholipid via a cofactor, ß2-glycoprotein 1 (ß2GP1); these antibodies showed excellent specificity for tumor vasculature and induce an immune stimulatory environment. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) constructed by linking PS-binding domain V of ß2GP1 to the Fc of an IgG2a. Betabodies bind to externalized PS with high affinity (∼1 nM), without the requirement of a co-factor and localize robustly to the TME. We demonstrate that betabodies are a direct PS-targeting agent that has the potential to be used as anti-tumor therapy, drug delivery vehicles, and tools for imaging the TME.

Molecular insights into human phosphatidylserine synthase 1 reveal its inhibition promotes LDL uptake.

In mammalian cells, two phosphatidylserine (PS) synthases drive PS synthesis. Gain-of-function mutations in the Ptdss1 gene lead to heightened PS production, causing Lenz-Majewski syndrome (LMS). Recently, pharmacological inhibition of PSS1 has been shown to suppress tumorigenesis. Here, we report the cryo-EM structures of wild-type human PSS1 (PSS1WT), the LMS-causing Pro269Ser mutant (PSS1P269S), and PSS1WT in complex with its inhibitor DS55980254. PSS1 contains 10 transmembrane helices (TMs), with TMs 4-8 forming a catalytic core in the luminal leaflet. These structures revealed a working mechanism of PSS1 akin to the postulated mechanisms of the membrane-bound O-acyltransferase family. Additionally, we showed that both PS and DS55980254 can allosterically inhibit PSS1 and that inhibition by DS55980254 activates the SREBP pathways, thus enhancing the expression of LDL receptors and increasing cellular LDL uptake. This work uncovers a mechanism of mammalian PS synthesis and suggests that selective PSS1 inhibitors have the potential to lower blood cholesterol levels.

Collagen patches releasing phosphatidylserine liposomes guide M1-to-M2 macrophage polarization and accelerate simultaneous bone and muscle healing.

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. STATEMENT OF SIGNIFICANCE: 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.

Optimal development of apoptotic cells-mimicking liposomes targeting macrophages.

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.

Under Heparin-Free Conditions Unsaturated Phospholipids Inhibit the Aggregation of 1N4R and 2N4R Tau.

A progressive aggregation of Tau proteins in the brain is linked to both Alzheimer's disease (AD) and various Tauopathies. This pathological process can be enhanced by several substances, including heparin. However, very little if anything is known about molecules that can inhibit the aggregation of Tau isoforms. In this study, we examined the effect of phosphatidylserines (PSs) with various lengths and saturations of fatty acids (FAs) on the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N-terminal inserts that enhance binding of Tau to tubulin. We found that PS with unsaturated and short-length FAs inhibited Tau aggregation and drastically lowered the toxicity of Tau oligomers that were formed in the presence of such phospholipids. Such an effect was not observed for PS with fully saturated long-chain FAs. These results suggest that a short-chain irreversible disbalance between saturated and unsaturated lipids in the brain could be the trigger of Tau aggregation.

Uncovering key steps in FGF12 cellular release reveals a common mechanism for unconventional FGF protein secretion.

FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long "a" isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short "b" isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins.