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ACS Biomaterials Science & Engineering May 2024Macrophage uptake of nanoparticles is highly dependent on the physicochemical characteristics of those nanoparticles. Here, we have created a collection of lipid-polymer...
Macrophage uptake of nanoparticles is highly dependent on the physicochemical characteristics of those nanoparticles. Here, we have created a collection of lipid-polymer nanoparticles (LPNPs) varying in size, stiffness, and lipid makeup to determine the effects of these factors on uptake in murine bone marrow-derived macrophages. The LPNPs varied in diameter from 232 to 812 nm, in storage modulus from 21.2 to 287 kPa, and in phosphatidylserine content from 0 to 20%. Stiff, large nanoparticles with a coating containing phosphatidylserine were taken up by macrophages to a much higher degree than any other formulation (between 9.3× and 166× higher than other LPNPs). LPNPs with phosphatidylserine were taken up most by M2-polarized macrophages, while those without were taken up most by M1-polarized macrophages. Differences in total LPNP uptake were not dependent on endocytosis pathway(s) other than phagocytosis. This work acts as a basis for understanding how the interactions between nanoparticle physicochemical characteristics may act synergistically to facilitate particle uptake.
Topics: Nanoparticles; Animals; Macrophages; Mice; Polymers; Lipids; Particle Size; Phagocytosis; Endocytosis; Phosphatidylserines
PubMed: 38657240
DOI: 10.1021/acsbiomaterials.3c01704 -
The Journal of Clinical Investigation Apr 2024BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies... (Clinical Trial)
Clinical Trial
BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-β-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.
Topics: Humans; Malaria, Cerebral; Autoantibodies; Female; Male; Platelet Factor 4; Child; Child, Preschool; Infant; Polyelectrolytes; Thrombosis
PubMed: 38652559
DOI: 10.1172/JCI176466 -
The Journal of Clinical Investigation Apr 2024Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to...
Carbohydrates and lipids provide the majority of substrates to fuel mitochondrial oxidative phosphorylation. Metabolic inflexibility, defined as an impaired ability to switch between these fuels, is implicated in a number of metabolic diseases. Here, we explore the mechanism by which physical inactivity promotes metabolic inflexibility in skeletal muscle. We developed a mouse model of sedentariness, small mouse cage (SMC), that, unlike other classic models of disuse in mice, faithfully recapitulated metabolic responses that occur in humans. Bioenergetic phenotyping of skeletal muscle mitochondria displayed metabolic inflexibility induced by physical inactivity, demonstrated by a reduction in pyruvate-stimulated respiration (JO2) in the absence of a change in palmitate-stimulated JO2. Pyruvate resistance in these mitochondria was likely driven by a decrease in phosphatidylethanolamine (PE) abundance in the mitochondrial membrane. Reduction in mitochondrial PE by heterozygous deletion of phosphatidylserine decarboxylase (PSD) was sufficient to induce metabolic inflexibility measured at the whole-body level, as well as at the level of skeletal muscle mitochondria. Low mitochondrial PE in C2C12 myotubes was sufficient to increase glucose flux toward lactate. We further implicate that resistance to pyruvate metabolism is due to attenuated mitochondrial entry via mitochondrial pyruvate carrier (MPC). These findings suggest a mechanism by which mitochondrial PE directly regulates MPC activity to modulate metabolic flexibility in mice.
Topics: Animals; Mice; Muscle, Skeletal; Pyruvic Acid; Mitochondria, Muscle; Phosphatidylethanolamines; Sedentary Behavior; Male; Carboxy-Lyases; Mice, Knockout; Stearoyl-CoA Desaturase
PubMed: 38652544
DOI: 10.1172/JCI167371 -
Molecular Pharmaceutics May 2024Amyloid oligomers and fibrils are protein aggregates that exert a high cell toxicity. Efficient degradation of these protein aggregates can minimize the spread and...
Amyloid oligomers and fibrils are protein aggregates that exert a high cell toxicity. Efficient degradation of these protein aggregates can minimize the spread and progression of neurodegeneration. In this study, we investigate the properties of natural killer (NK) cells and macrophages in the degradation of α-synuclein (α-Syn) aggregates grown in a lipid-free environment and in the presence of phosphatidylserine and cholesterol (PS/Cho), which are lipids that are directly associated with the onset and progression of Parkinson's disease. We found that both types of α-Syn aggregates were endocytosed by neurons, which caused strong damage to cell endosomes. Our results also indicated that PS/Cho vesicles drastically increased the toxicity of α-Syn fibrils formed in their presence compared to the toxicity of α-Syn aggregates grown in a lipid-free environment. Both NK cells and macrophages were able to degrade α-Syn and α-Syn/Cho monomers, oligomers, and fibrils. Quantitative analysis of protein degradation showed that macrophages demonstrated substantially more efficient internalization and degradation of amyloid aggregates in comparison to NK cells. We also found that amyloid aggregates induced the proliferation of macrophages and NK cells and significantly changed the expression of their cytokines and chemokines.
Topics: alpha-Synuclein; Macrophages; Killer Cells, Natural; Humans; Amyloid; Protein Aggregates; Animals; Mice; Cholesterol; Phosphatidylserines; Parkinson Disease; Neurons; Endocytosis; Cell Proliferation; Cytokines
PubMed: 38635186
DOI: 10.1021/acs.molpharmaceut.4c00160 -
The EMBO Journal May 2024Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly...
Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P levels, the recovery of the PM pool of PI(4,5)P after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape.
Topics: Humans; Phosphatidylinositols; Phospholipid Transfer Proteins; Lipid Metabolism; Cell Membrane; HeLa Cells; Organelles; Endosomes; Animals
PubMed: 38627600
DOI: 10.1038/s44318-024-00096-3 -
Cell Reports Apr 2024Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4 large peritoneal...
Receptors controlling the cross-presentation of tumor antigens by macrophage subsets in cancer tissues are poorly explored. Here, we show that TIM4 large peritoneal macrophages efficiently capture and cross-present tumor-associated antigens at early stages of peritoneal infiltration by ovarian cancer cells. The phosphatidylserine (PS) receptor TIM4 promotes maximal uptake of dead cells or PS-coated artificial targets and triggers inflammatory and metabolic gene programs in combination with cytoskeletal remodeling and upregulation of transcriptional signatures related to antigen processing. At the cellular level, TIM4-mediated engulfment induces nucleation of F-actin around nascent phagosomes, delaying the recruitment of vacuolar ATPase, acidification, and cargo degradation. In vivo, TIM4 deletion blunts induction of early anti-tumoral effector CD8 T cells and accelerates the progression of ovarian tumors. We conclude that TIM4-mediated uptake drives the formation of specialized phagosomes that prolong the integrity of ingested antigens and facilitate cross-presentation, contributing to immune surveillance of the peritoneum.
Topics: Animals; Macrophages, Peritoneal; Female; Mice; Carcinogenesis; Humans; Antigens, Neoplasm; Ovarian Neoplasms; Membrane Proteins; Mice, Inbred C57BL; Cross-Priming; Cell Line, Tumor; Phagosomes; Antigen Presentation; CD8-Positive T-Lymphocytes; Actins
PubMed: 38607919
DOI: 10.1016/j.celrep.2024.114096 -
MBio May 2024Systemic infections by spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug...
UNLABELLED
Systemic infections by 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 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 nucleotidase-coupled malachite-green-based high throughput assay for purified 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 ' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting Cho1 function by inducing phenotypes consistent with the 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 of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.
IMPORTANCE
Fungal 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 value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.
Topics: Candida albicans; Antifungal Agents; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Enzyme Inhibitors; High-Throughput Screening Assays; Small Molecule Libraries; Microbial Sensitivity Tests; Fungal Proteins; Phosphatidylserines; Furans; Thiophenes
PubMed: 38587428
DOI: 10.1128/mbio.00633-24 -
Inhibition of malaria and babesiosis parasites by putative red blood cell targeting small molecules.Frontiers in Cellular and Infection... 2024Chemotherapies for malaria and babesiosis frequently succumb to the emergence of pathogen-related drug-resistance. Host-targeted therapies are thought to be less...
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 infection. Alongside , we implemented parasite susceptibility assays also in the zoonotic parasite and the veterinary parasite . 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 -specific, specific, and broad-spectrum antiparasitic activity. The anticancer MEK-targeting drug trametinib is shown here to act with submicromolar activity to block proliferation of 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.
Topics: Animals; Humans; Parasites; Babesiosis; Malaria; Erythrocytes; Plasmodium; Malaria, Falciparum; Babesia; Antimalarials; Plasmodium falciparum
PubMed: 38572319
DOI: 10.3389/fcimb.2024.1304839 -
Acta Pharmaceutica Sinica. B Apr 2024In the treatment of central nervous system disease, the blood-brain barrier (BBB) is a major obstruction to drug delivery that must be overcome. In this study, we...
In the treatment of central nervous system disease, the blood-brain barrier (BBB) is a major obstruction to drug delivery that must be overcome. In this study, we propose a brain-targeted delivery strategy based on selective opening of the BBB. This strategy allows some simple bare nanoparticles to enter the brain when mixed with special opening material; however, the BBB still maintains the ability to completely block molecules from passing through. Based on the screening of BBB opening and matrix delivery materials, we determined that phospholipase A2-catalyzed 1-palmitoyl-2-oleoyl--glycero-3-phosphoserine liposomes can efficiently carry drugs into the brain immediately. At an effective dose, this delivery system is safe, especially with its effect on the BBB being reversible. This mix & act delivery system has a simple structure and rapid preparation, making it a strong potential candidate for drug delivery across the BBB.
PubMed: 38572103
DOI: 10.1016/j.apsb.2023.11.015 -
Tobacco Induced Diseases 2024The use of e-cigarettes (ECs) has reached unprecedented levels, due to a variety of reasons, including the misconception regarding their safety. Thus, there have been...
INTRODUCTION
The use of e-cigarettes (ECs) has reached unprecedented levels, due to a variety of reasons, including the misconception regarding their safety. Thus, there have been efforts to characterize the effects of EC exposure, including in the context of thirdhand EC (THEC) on a host of disorders, such as cardiovascular disease (CVD).
METHODS
To address this issue, we sought to characterize the effects of THEC on platelet function and thrombus formation, using a novel mouse exposure protocol that resembles real life scenarios. To assess these effects, a host of related (i.e. tail bleeding time, and ferric chloride injury induced thrombosis model) assays and platelet specific (e.g. aggregation, and dense granule secretion) investigative assays were conducted.
RESULTS
Our characterization demonstrated that THEC exposed mice exhibited a prothrombotic phenotype reflected by their shortened tail bleeding (THEC: 37 ± 15 seconds, versus clean air: 183 ± 56 s) and occlusion times (THEC: 188 ± 39 s, versus clean air: 519 ± 70 s), relative to those exposed to clean air. Importantly, we found no difference in the platelet counts between the THEC and clean air mice. As for the underlying mechanism, separate experiments revealed significantly enhanced platelet aggregation, dense and alpha granule secretion, as well as integrin/GPIIb-IIIa activation and phosphatidylserine exposure in response to thrombin and ADP agonist stimulation.
CONCLUSIONS
Taken together, these results provide evidence that THEC does have the capacity to increase the risk of thrombotic disease, which should increase awareness regarding its underappreciated negative health effects.
PubMed: 38560550
DOI: 10.18332/tid/185286