Macrophage Polarization Status Impacts Nanoceria Cellular Distribution but Not Its Biotransformation or Ferritin Effects

The innate immune system is the first line of defense against external threats through the initiation and regulation of inflammation. Macrophage differentiation into functional pheno- types influences the fate of nanomaterials taken up by these immune cells. High-resolution electron microscopy was used to investigate the uptake, distribution, and biotransformation of nanoceria in human and murine M1 and M2 macrophages in unprecedented detail. We found that M1 and M2 macrophages internalize nanoceria differently. M1-type macrophages predominantly sequester nanoceria near the plasma membrane, whereas nanoceria are more uniformly distributed throughout M2 macrophage cytoplasm. In contrast, both macrophage phenotypes show identical nanoceria biotransformation to cerium phosphate nanoneedles and simultaneous nanoceria with ferritin co- precipitation within the cells. Ferritin biomineralization is a direct response to nanoparticle uptake inside both macrophage phenotypes. We also found that the same ferritin biomineralization mecha- nism occurs after the uptake of Ce-ions into polarized macrophages and into unpolarized human monocytes and murine RAW 264.7 cells. These findings emphasize the need for evaluating ferritin biomineralization in studies that involve the internalization of nano objects, ranging from particles to viruses to biomolecules, to gain greater mechanistic insights into the overall immune responses to nano object

Role of Leukotriene B4 Receptors in the Development of Atherosclerosis: Potential Mechanisms

Objective-Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB 4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-/- mice. Methods and Results-Analysis of global changes in gene expression induced by LTB 4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2, which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4. Conclusions-The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation

Brain Microvascular Endothelial Cell Association and Distribution of a 5 nm Ceria Engineered Nanomaterial

PURPOSE: Ceria engineered nanomaterials (ENMs) have current commercial applications and both neuroprotective and toxic effects. Our hypothesis is that ceria ENMs can associate with brain capillary cells and/or cross the blood-brain barrier. METHODS: An aqueous dispersion of ∼5 nm ceria ENM was synthesized and characterized in house. Its uptake space in the Sprague Dawley rat brain was determined using the in situ brain perfusion technique at 15 and 20 mL/minute flow rates; 30, 100, and 500 μg/mL ceria perfused for 120 seconds at 20 mL/minute; and 30 μg/mL perfused for 20, 60, and 120 seconds at 20 mL/minute. The capillary depletion method and light and electron microscopy were used to determine its capillary cell and brain parenchymal association and localization. RESULTS: The vascular space was not significantly affected by brain perfusion flow rate or ENM, demonstrating that this ceria ENM did not influence blood-brain barrier integrity. Cerium concentrations, determined by inductively coupled plasma mass spectrometry, were significantly higher in the choroid plexus than in eight brain regions in the 100 and 500 μg/mL ceria perfusion groups. Ceria uptake into the eight brain regions was similar after 120-second perfusion of 30, 100, and 500 μg ceria/mL. Ceria uptake space significantly increased in the eight brain regions and choroid plexus after 60 versus 20 seconds, and it was similar after 60 and 120 seconds. The capillary depletion method showed 99.4% ± 1.1% of the ceria ENM associated with the capillary fraction. Electron microscopy showed the ceria ENM located on the endothelial cell luminal surface. CONCLUSION: Ceria ENM association with brain capillary endothelial cells saturated between 20 and 60 seconds and ceria ENM brain uptake was not diffusion-mediated. During the 120-second ceria ENM perfusion, ceria ENM predominately associated with the surface of the brain capillary cells, providing the opportunity for its cell uptake or redistribution back into circulating blood

Biodistribution and Biopersistence of Ceria Engineered Nanomaterials: Size Dependence

The aims were to determine the biodistribution, translocation, and persistence of nanoceria in the brain and selected peripheral organs. Nanoceria is being studied as an anti-oxidant therapeutic. Five, 15, 30, or 55 nm ceria was iv infused into rats which were terminated 1, 20, or 720 h later. Cerium was determined in blood, brain, liver, and spleen. Liver and spleen contained a large percentage of the dose, from which there was no significant clearance over 720 h, associated with adverse changes. Very little nanoceria entered brain parenchyma. The results suggest brain delivery of nanoceria will be a challenge. FROM THE CLINICAL EDITOR: This team of investigators revealed that nanoceria, which is being studied as an anti-oxidant, has very limited uptake by the brain regardless of the range of sizes studied, suggesting major challenges in the application of this novel approach in the central nervous system

The Anti-Sigma Factor MucA of Pseudomonas aeruginosa: Dramatic Differences of a mucA22 vs. a ΔmucA Mutant in Anaerobic Acidified Nitrite Sensitivity of Planktonic and Biofilm Bacteria in vitro and During Chronic Murine Lung Infection

Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157–194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases

Analytical High-Resolution Electron Microscopy Reveals Organ-Specific Nanoceria Bioprocessing

This is the first utilization of advanced analytical electron microscopy methods, including high-resolution transmission electron microscopy, high-angle annular dark field scanning transmission electron microscopy, electron energy loss spectroscopy, and energy-dispersive X-ray spectroscopy mapping to characterize the organ-specific bioprocessing of a relatively inert nanomaterial (nanoceria). Liver and spleen samples from rats given a single intravenous infusion of nanoceria were obtained after prolonged (90 days) in vivo exposure. These advanced analytical electron microscopy methods were applied to elucidate the organ-specific cellular and subcellular fate of nanoceria after its uptake. Nanoceria is bioprocessed differently in the spleen than in the liver

Cerium dioxide, a Jekyll and Hyde nanomaterial, can increase basal and decrease elevated inflammation and oxidative stress

It was hypothesized that the catalyst nanoceria can increase oxidative stress/inflammation from the basal state and reduce it from the elevated state . Nanoceria are cleared by macrophages. To test the hypothesis, M0 (non-polarized), M1- (classically activated, pro-inflammatory), and M2-like (alternatively activated, regulatory phenotype) RAW 264.7 macrophages were nanoceria exposed. Responses were quantified by arginase activity, IL-1ß level, cell oxygen consumption rate (OCR), the glycolysis stress test (GST), morphology determined by light microscopy, macrophage phenotype marker expression and morphology using a novel three dimensional immunohistochemical method, and RT-qPCR. Nanoceria blocked arginase and IL-1ß effects, increased M0 cell OCR and GST toward the M2 phenotype and altered multiple M1- and M2-like cell endpoints toward the M0 level. M1-like cells had greater volume and less circularity/roundness, and the M2-like cells had greater volume than M0 macrophages. Nanoceria converted M1- and M2-like cells toward M0 morphology. The results are overall consistent with the hypothesis

Adversarial Policies Beat Superhuman Go AIs

We attack the state-of-the-art Go-playing AI system KataGo by training adversarial policies against it, achieving a >97% win rate against KataGo running at superhuman settings. Our adversaries do not win by playing Go well. Instead, they trick KataGo into making serious blunders. Our attack transfers zero-shot to other superhuman Go-playing AIs, and is comprehensible to the extent that human experts can implement it without algorithmic assistance to consistently beat superhuman AIs. The core vulnerability uncovered by our attack persists even in KataGo agents adversarially trained to defend against our attack. Our results demonstrate that even superhuman AI systems may harbor surprising failure modes. Example games are available https://goattack.far.ai/.Comment: Accepted to ICML 2023, see paper for changelo

An expanded toolkit for gene tagging based on MiMIC and scarless CRISPR tagging in

We generated two new genetic tools to efficiently tag genes in Drosophila. The first, Double Header (DH) utilizes intronic MiMIC/CRIMIC insertions to generate artificial exons for GFP mediated protein trapping or T2A-GAL4 gene trapping in vivo based on Cre recombinase to avoid embryo injections. DH significantly increases integration efficiency compared to previous strategies and faithfully reports the expression pattern of genes and proteins. The second technique targets genes lacking coding introns using a two-step cassette exchange. First, we replace the endogenous gene with an excisable compact dominant marker using CRISPR making a null allele. Second, the insertion is replaced with a protein::tag cassette. This sequential manipulation allows the generation of numerous tagged alleles or insertion of other DNA fragments that facilitates multiple downstream applications. Both techniques allow precise gene manipulation and facilitate detection of gene expression, protein localization and assessment of protein function, as well as numerous other applications. Research organism: D. melanogaste

From Dose to Response: In Vivo Nanoparticle Processing and Potential Toxicity

Adverse human health impacts due to occupational and environmental exposures to manufactured nanoparticles are of concern and pose a potential threat to the continued industrial use and integration of nanomaterials into commercial products. This chapter addresses the inter-relationship between dose and response and will elucidate on how the dynamic chemical and physical transformation and breakdown of the nanoparticles at the cellular and subcellular levels can lead to the in vivo formation of new reaction products. The dose-response relationship is complicated by the continuous physicochemical transformations in the nanoparticles induced by the dynamics of the biological system, where dose, bio-processing, and response are related in a non-linear manner. Nanoscale alterations are monitored using high-resolution imaging combined with in situ elemental analysis and emphasis is placed on the importance of the precision of characterization. The result is an in-depth understanding of the starting particles, the particle transformation in a biological environment, and the physiological response