Search

IntroductionOxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of particulate matter with aerodynamic diameter ≤2.5 µm (PM2.5). PM2.5 inhalation and deposition into the respiratory tract causes the formation of ROS by chemical reactions and phagocytosis of macrophages in the epithelial lining fluid (ELF), but their relative contributions are not well quantified and their link to oxidative stress remains uncertain. The specific aims of this project were (1) elucidating the chemical mechanism and quantifying the formation kinetics of ROS in the ELF by SOA; (2) quantifying the relative importance of ROS formation by chemical reactions and macrophages in the ELF.MethodsSOA particles were generated using reaction chambers from oxidation of various precursors including isoprene, terpenes, and aromatic compounds with or without nitrogen oxides (NOx). We collected size-segregated PM at two highway sites in Anaheim, CA, and Long Beach, CA, and at an urban site in Irvine, CA, during two wildfire events. The collected particles were extracted into water or surrogate ELF that contained lung antioxidants. ROS generation was quantified using electron paramagnetic resonance (EPR) spectroscopy with a spin-trapping technique. PM oxidative potential (OP) was also quantified using the dithiothreitol assay. In addition, kinetic modeling was applied for analysis and interpretation of experimental data. Finally, we quantified cellular superoxide release by RAW264.7 macrophage cells upon exposure to quinones and isoprene SOA using a chemiluminescence assay as calibrated with an EPR spin-probing technique. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes.ResultsSuperoxide radicals (·O2-) were formed from aqueous reactions of biogenic SOA generated by hydroxy radical (·OH) photooxidation of isoprene, β-pinene, α-terpineol, and d-limonene. The temporal evolution of ·OH and ·O2- formation was elucidated by kinetic modeling with a cascade of aqueous reactions, including the decomposition of organic hydroperoxides (ROOH), ·OH oxidation of primary or secondary alcohols, and unimolecular decomposition of α-hydroxyperoxyl radicals. Relative yields of various types of ROS reflected the relative abundance of ROOH and alcohols contained in SOA, which generated under high NOx conditions, exhibited lower ROS yields. ROS formation by SOA was also affected by pH. Isoprene SOA had higher ·OH and organic radical yields at neutral than at acidic pH. At low pH ·O2- was the dominant species generated by all types of SOA. At neutral pH, α-terpineol SOA exhibited a substantial yield of carbon-centered organic radicals (R·), while no radical formation was observed by aromatic SOA.Organic radicals in the ELF were formed by mixtures of Fe2+ and SOA generated from photooxidation of isoprene, α-terpineol, and toluene. The molar yields of organic radicals by SOA were 5-10 times higher in ELF than in water. Fe2+ enhanced organic radical yields by a factor of 20-80. Ascorbate mediated redox cycling of iron ions and sustained organic peroxide decomposition, as supported by kinetic modeling reproducing time- and concentration-dependence of organic radical formation, as well as by additional experiments observing the formation of Fe2+ and ascorbate radicals in mixtures of ascorbate and Fe3+. ·OH and superoxide were found to be efficiently scavenged by antioxidants.Wildfire PM mainly generated ·OH and R· with minor contributions from superoxide and oxygen-centered organic radicals (RO·). PM OP was high in wildfire PM, exhibiting very weak correlation with radical forms of ROS. These results were in stark contrast with PM collected at highway and urban sites, which generated much higher amounts of radicals dominated by ·OH radicals that correlated well with OP. By combining field measurements of size-segregated chemical composition, a human respiratory tract model, and kinetic modeling, we quantified production rates and concentrations of different types of ROS in different regions of the ELF by considering particle-size-dependent respiratory deposition. While hydrogen peroxide (H2O2) and ·O2- production were governed by Fe and Cu ions, ·OH radicals were mainly generated by organic compounds and Fenton-like reactions of metal ions. We obtained mixed results for correlations between PM OP and ROS formation, providing rationale and limitations of the use of oxidative potential as an indicator for PM toxicity in epidemiological and toxicological studies.Quinones and isoprene SOA activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in macrophages, releasing massive amounts of superoxide via respiratory burst and overwhelming the superoxide formation by aqueous chemical reactions in the ELF. The threshold dose for macrophage activation was much smaller for quinones compared with isoprene SOA. The released ROS caused lipid peroxidation to increase cell membrane fluidity, inducing oxidative damage and stress. Further increases of doses led to the activation of antioxidant response elements, reducing the net cellular superoxide production. At very high doses and long exposure times, chemical production became comparably important or dominant if the escalation of oxidative stress led to cell death.ConclusionsThe mechanistic understandings and quantitative information on ROS generation by SOA particles provided a basis for further elucidation of adverse aerosol health effects and oxidative stress by PM2.5. For a comprehensive assessment of PM toxicity and health effects via oxidative stress, it is important to consider both chemical reactions and cellular processes for the formation of ROS in the ELF. Chemical composition of PM strongly influences ROS formation; further investigations are required to study ROS formation from various PM sources. Such research will provide critical information to environmental agencies and policymakers for the development of air quality policy and regulation.

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, a red macroalga, is a commercial source of carrageenan, a widely used polysaccharide compound important in the food and pharmaceutical industries, in nanotechnology, and in pharmacological applications. Carrageenan is found mainly in the cell wall and in the intercellular matrix. This is the first study to propose the characterization of carrageenans in vitro, using the auto-fluorescence properties of the alga treated with different polyamines: putrescine, spermidine, and spermine. This study suggests a four-phase cultivation sequence for seaweed farmers to enhance and assess the potential carrageenan yield of their crops. In phase 1, seedlings were treated with each of the polyamines. Explants were subsequently transferred through two additional culture phases before being planted on the sea farms in phase 4 and then harvested after 60 days for analysis. Images from transverse sections of 11 representative cultured K. alvarezii samples were obtained at 561 nm excitation wavelength for both the cell center and the cell wall of each sample. Spectral data were also analyzed using the spectral phasor algorithm of SimFCS developed at the Laboratory for Fluorescence Dynamics (www.lfd.uci.edu). We report on the identification of several spectral fluorescence emission fingerprints from different auto-fluorescence compounds spatially mapped using this technique. These fingerprints have the potential to improve strain selection of explants for enhanced carrageenan yield in seaweed farming operations as well as to enable wholesale pricing to correspond with crop quality.

Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.

© OSA 2015. Detecting proteins dynamics within cells grown in 3D micro-environments is challenging. We developed a 3D nano-imaging technique to uniquely probes proteins in cells grown on collagen. Results show paxillin and actin diffusion rates are unique.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

We report herein a platform technology called 'microencapsulated sensors' that is able to selectively detect bacteria in unprocessed whole blood at single-cell sensitivity in a one-step, homogenous, and culture- and amplification-free reaction. Our system integrates real-time DNAzyme sensor technology and droplet microfluidics.

Fluorescence spectroscopy is the most common non-radioactive technique used to study GPCR interactions with their ligands. Raster image correlation spectroscopy (RICS) exploits spatio-temporal correlation functions rather than the simple temporal correlations of conventional fluorescence correlation spectroscopy. In this paper we describe the use of RICS and the number and brightness method to determine the diffusion of a construct of endothelin ETA receptor with EGFP and the aggregation state in the cytoplasm. Our construct seems to locate mainly in the cytoplasm where it undergoes diffusion and it appears to be monomeric. Although our construct could not fully represent the native protein, we believe that the methodology we describe in this paper could be used by anyone in this field.

Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 µm2/s). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity ν ≈ 0.03 µm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.

We present a simple, ultra-rapid and robust method to create sharp nanostructures-nanopetals-in a shape memory polymer substrate demonstrating unprecedented enhancements for surface enhanced sensing over large surface areas. These bimetallic nanostructures demonstrate extremely strong surface plasmon resonance effects due to the high density multifaceted petal structures that increase the probability of forming nanogaps. We demonstrate that our nanopetals exhibit extremely strong surface plasmons, confining the emission and enhancing the fluorescence intensity of the nearby high-quantum yield fluorescein by >4000×. The enhancements are confined to the extremely small volumes at the nanopetal borders. This enables us to achieve single molecule detection at relatively high and physiological concentrations. © 2010 American Institute of Physics.

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as approximately 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin-enhanced-green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.

To combat the rise of antibiotic-resistance in bacteria and the resulting effects on healthcare worldwide, new technologies are needed that can perform rapid antibiotic susceptibility testing (AST). Conventional clinical methods for AST rely on growth-based assays, which typically require long incubation times to obtain quantitative results, representing a major bottleneck in the determination of the optimal antibiotic regimen to treat patients. Here, we demonstrate a rapid AST method based on the metabolic activity measured by fluorescence lifetime imaging microscopy (FLIM). Using lab strains and clinical isolates of Escherichia coli with tetracycline-susceptible and resistant phenotypes as models, we demonstrate that changes in metabolic state associated with antibiotic susceptibility can be quantitatively tracked by FLIM. Our results show that the magnitude of metabolic perturbation resulting from antibiotic activity correlates with susceptibility evaluated by conventional metrics. Moreover, susceptible and resistant phenotypes can be differentiated in as short as 10 min after antibiotic exposure. This FLIM-AST (FAST) method can be applied to other antibiotics and provides insights into the nature of metabolic perturbations inside bacterial cells resulting from antibiotic exposure with single cell resolution.

Mutations in dynamin 2 (DNM2) have been associated with two distinct movement disorders: Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). Most of these mutations are clustered in the pleckstrin homology domain (PHD), which engages in intramolecular interactions that limit dynamin self-assembly and GTPase activation. CNM mutations interfere with these intramolecular interactions and suppress the formation of the autoinhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for phosphoinositide binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations causes either one disease or the other, despite their close proximity within the PHD sequence. We previously reported that at least one CMT-causing mutant, lacking residues 555 DEE 557 (ΔDEE), displays the same inability to undergo autoinhibition as observed in CNM-linked mutants. Here, we show that both the DNM2 ΔDEE and CNM-linked DNM2 A618T mutants form larger and more stable structures on the plasma membrane than that of wild-type DNM2 (DNM2 WT ). However, DNM2 A618T forms cytoplasmic inclusions at concentrations lower than those of either DNM2 WT or DNM2 ΔDEE , suggesting that CNM-linked mutations confer more severe gain-of-function properties than the ΔDEE mutation.

Bioluminescence is a powerful method for imaging in vivo , but applications at the microscale are far from routine. This is due, in part, to a lack of versatile tools for visualizing dynamic events. To address this void, we developed a new platform-Bioluminescence Resonance Energy mAKe over with a Fluorescence-Activating absorption-Shifting Tag (BREAKFAST). BREAKFAST features a bright luciferase combined with a chemogenetic tag (pFAST) for rapid color switching. In the presence of luciferin and a discrete fluorogenic ligand, signal is observed via resonance energy transfer. We evaluated spectral outputs with various fluorogens and established the utility of BREAKFAST for combined fluorescence and bioluminescence imaging. Dynamic, four-color visualization was achieved with sequential ligand addition and spectral phasor analysis. We further showed selective signal quenching with a dark fluorogen. Collectively, this work establishes a new method for bioluminescence imaging at the cellular scale and sets the stage for continued probe development., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (This journal is © The Royal Society of Chemistry.)

Biomaterial wound dressings, such as hydrogels, interact with host cells to regulate tissue repair. This study investigates how crosslinking of gelatin-based hydrogels influences immune and stromal cell behavior and wound healing in female mice. We observe that softer, lightly crosslinked hydrogels promote greater cellular infiltration and result in smaller scars compared to stiffer, heavily crosslinked hydrogels. Using single-cell RNA sequencing, we further show that heavily crosslinked hydrogels increase inflammation and lead to the formation of a distinct macrophage subpopulation exhibiting signs of oxidative activity and cell fusion. Conversely, lightly crosslinked hydrogels are more readily taken up by macrophages and integrated within the tissue. The physical properties differentially affect macrophage and fibroblast interactions, with heavily crosslinked hydrogels promoting pro-fibrotic fibroblast activity that drives macrophage fusion through RANKL signaling. These findings suggest that tuning the physical properties of hydrogels can guide cellular responses and improve healing, offering insights for designing better biomaterials for wound treatment., (© 2024. The Author(s).)

We implemented a multimodal set of functional imaging techniques optimized for deep-tissue imaging to investigate how cancer cells invade surrounding tissues and how their physiological properties change in the process. As a model for cancer invasion of the extracellular matrix, we created 3D spheroids from triple-negative breast cancer cells (MDA-MB-231) and non-tumorigenic breast epithelial cells (MCF-10A). We analyzed multiple hallmarks of cancer within the same spheroid by combining a number of imaging techniques, such as metabolic imaging of NADH by Fluorescence Lifetime Imaging Microscopy (NADH-FLIM), hyperspectral imaging of a solvatochromic lipophilic dye (Nile Red) and extracellular matrix imaging by Second Harmonic Generation (SHG). We included phasor-based bioimage analysis of spheroids at three different time points, tracking both morphological and biological properties, including cellular metabolism, fatty acids storage, and collagen organization. Employing this multimodal deep-imaging framework, we observed and quantified cancer cell plasticity in response to changes in the environment composition.

Macrophages exhibit a spectrum of behaviors upon activation and are generally classified as one of two types: inflammatory (M1) or anti-inflammatory (M2). Tracking these phenotypes in living cells can provide insight into immune function, but remains a challenging pursuit. Existing methods are mostly limited to static readouts or difficult to employ for multiplexed imaging in complex 3D environments while maintaining cellular resolution. We aimed to fill this void using bioluminescent technologies. Here we report genetically engineered luciferase reporters for long-term monitoring of macrophage polarization via spectral phasor analysis. M1- and M2- specific promoters were used to drive the expression of bioluminescent enzymes in macrophage cell lines. The readouts were multiplexed and discernable in both 2D and 3D formats with single cell resolution in living samples. Collectively, this work expands the toolbox of methods for monitoring macrophage polarization and provides a blueprint for monitoring other multifaceted networks in heterogeneous environments.