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Introduction: Oxidative 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 (PM 2.5 ). PM 2.5 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., Methods: SOA particles were generated using reaction chambers from oxidation of various precursors including isoprene, terpenes, and aromatic compounds with or without nitrogen oxides (NO x ). 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., Results: Superoxide radicals (·O 2 - ) 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 ·O 2 - 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 NO x 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 ·O 2 - 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 Fe 2+ 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. Fe 2+ 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 Fe 2+ and ascorbate radicals in mixtures of ascorbate and Fe 3+ . ·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 (H 2 O 2 ) and ·O 2 - 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., Conclusions: The 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 PM 2.5 . 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., (© 2023 Health Effects Institute. All rights reserved.)

The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core. We then developed 3D scaffolds with μ-compressive moduli resembling either stiffer tumor core or softer peritumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis, which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of fluorophore species contributing to the signal is unknown, while excited state lifetimes with <1 ns difference are difficult to discriminate. Finally, existing analyses require high photon budgets and often cannot rigorously propagate experimental uncertainty into values over lifetime maps and number of species involved. To overcome all of these challenges simultaneously and self-consistently at once, we propose the first doubly nonparametric framework. That is, we learn the number of species (using Beta-Bernoulli process priors) and absolute maps of these fluorophore species (using Gaussian process priors) by leveraging information from pulses not leading to observed photon. We benchmark our framework using a broad range of synthetic and experimental data and demonstrate its robustness across a number of scenarios including cases where we recover lifetime differences between species as small as 0.3 ns with merely 1000 photons., Competing Interests: The authors declare no competing financial interest.

Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes-outside the intermediate, Goldilocks, zone-for data drawn from a single confocal spot leveraging the tools of Bayesian nonparametrics (BNP). We benchmark our algorithm, termed BNP lifetime analysis, using a range of synthetic and experimental data. Moreover, we show that the BNP lifetime analysis method can distinguish and deduce lifetimes using photon counts as small as 500., Competing Interests: Declaration of interests Authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Oxidative 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 fine particulate matter, and their inhalation and deposition into the respiratory tract causes the formation of ROS by chemical and cellular processes, but their relative contributions are hardly quantified and their link to oxidative stress remains uncertain. Here, we quantified cellular and chemical superoxide generation by 9,10-phenanthrenequinone (PQN) and isoprene SOA using a chemiluminescence assay combined with electron paramagnetic resonance spectroscopy as well as kinetic modeling. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes. We show that PQN and isoprene SOA activate NADPH oxidase in macrophages to release massive amounts of superoxide, overwhelming the superoxide formation by aqueous chemical reactions in the epithelial lining fluid. The activation dose for PQN is 2 orders of magnitude lower than that of isoprene SOA, suggesting that quinones are more toxic. While higher exposures trigger cellular antioxidant response elements, the released ROS induce oxidative damage to the cell membrane through lipid peroxidation. Such mechanistic and quantitative understandings provide a basis for further elucidation of adverse health effects and oxidative stress by fine particulate matter.

The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial targeting with phenformin., (© 2022. The Author(s).)

An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate Apc- driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc , a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset cancer prevention.

Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time and over multiple length scales., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report direct blind unmixing of lifetimes with subnanosecond resolution and subpixel spatial resolution using standard raster scan FLIM images. We demonstrate our method using a wide range of simulated and experimental data., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of metastasis., (© 2021. The Author(s).)

UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers 1 . However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation 2-8 , the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity of MLL4. Moreover, UTX regulates genome-wide histone modifications and high-order chromatin interactions in a condensation-dependent manner. We also found that UTY, the Y chromosome homologue of UTX with weaker tumour-suppressive activity, forms condensates with reduced molecular dynamics. These studies demonstrate a crucial biological function of liquid condensates with proper material states in enabling the tumour-suppressive activity of a chromatin regulator., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding mitochondrial form and function., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K +  ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD, the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence imaging visualizes the absorption of the mutant Htt protein aggregates which results in a distinct NADH fluorescence lifetime between control cells and acceptor cells. These studies show, for the first time, the influence of how neighboring cells expressing the expanded Htt protein can regulate energy metabolism in healthy cells., (© 2021 IOP Publishing Ltd.)

The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was significantly faster in the apical membrane than in the basal membrane, suggesting diffusion hindrance by the adhesion of the cells to the substrate. This finding highlights the importance of selecting the appropriate membrane (i.e., basal or apical) and corresponding method when measuring receptor diffusion in live cells. Accurately knowing the diffusion coefficient of two macrophage receptors involved in the response to pathogen insults will facilitate the study of changes that occur in signaling in these cells as a result of aging and disease.

It remains unknown if biophysical or material properties of biomolecular condensates regulate cancer. Here we show that AKAP95, a nuclear protein that regulates transcription and RNA splicing, plays an important role in tumorigenesis by supporting cancer cell growth and suppressing oncogene-induced senescence. AKAP95 forms phase-separated and liquid-like condensates in vitro and in nucleus. Mutations of key residues to different amino acids perturb AKAP95 condensation in opposite directions. Importantly, the activity of AKAP95 in splice regulation is abolished by disruption of condensation, significantly impaired by hardening of condensates, and regained by substituting its condensation-mediating region with other condensation-mediating regions from irrelevant proteins. Moreover, the abilities of AKAP95 in regulating gene expression and supporting tumorigenesis require AKAP95 to form condensates with proper liquidity and dynamicity. These results link phase separation to tumorigenesis and uncover an important role of appropriate biophysical properties of protein condensates in gene regulation and cancer.

Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism to a higher fraction of bound NADH indicative of oxidative phosphorylation. The FLIM and viability results of MCF10A cells demonstrate that the treatments targeted cancer cells over the normal breast tissue. The identification of energy metabolism alteration could open up strategies of improving chemotherapy for malignant breast cancer.

The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD + concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total NAD(H) production and suggested the involvement of the glyoxylate pathway in virulence activation in surface-attached populations. In addition, we induced virulence at an earlier time using the electron transport chain oxidase inhibitor antimycin A. Our results demonstrate the use of FLIM to noninvasively measure NADH dynamics in biofilms and suggest a model in which a metabolic rearrangement accompanies the virulence activation period. IMPORTANCE The rise of antibiotic resistance requires the development of new strategies to combat bacterial infection and pathogenesis. A major direction has been the development of drugs that broadly target virulence. However, few targets have been identified due to the species-specific nature of many virulence regulators. The lack of a virulence regulator that is conserved across species has presented a further challenge to the development of therapeutics. Here, we identify that NADH activity has an important role in the induction of virulence in the pathogen P. aeruginosa This finding, coupled with the ubiquity of NADH in bacterial pathogens, opens up the possibility of targeting enzymes that process NADH as a potential broad antivirulence approach., (Copyright © 2020 Perinbam et al.)

Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast cancer.

Number and molecular Brightness (N&B) analysis is a powerful method used to monitor protein aggregation in living cells. Here, we used the N&B method to characterize the unexpanded HTT protein oligomerization after the internalization of the mutant HTT (mHTT) which contains a CAG repeat extensions encoding for long polyglutamine (polyQ) proteins resulting in misfolding and aggregation. HEK cells expressing Htt25Q-mCherry proteins were infected with Htt97Q-EGFP aggregates, by cell to cell uptake, in cultured conditions resulting in an increasing population of dimers and tetramers compared to our controls. This study shows for the first time the impact of protein aggregation in the unexpanded Htt25Q-mCherry expressing cells that occurs from cell to cell transfer of the expanded Htt97Q-EGFP. These results signify the sporadic behavior of the polyQ inclusion that gives insight into the mechanism of protein dynamics as a consequence of secreted mHTT aggregates., (Copyright © 2019. Published by Elsevier Inc.)

DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal the novel prosurvival response to PARP activation through a change in cellular metabolism and demonstrate how unique applications of advanced fluorescence imaging and laser microirradiation-induced DNA damage can be a powerful tool to interrogate damage-induced metabolic changes at high spatiotemporal resolution in a live cell.

Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime distribution patterns. We show, under our experimental conditions, that the phasor-FLIM approach provides a much-needed non-invasive quantitative technology for identifying healthy embryos at the early compaction stage with 86% accuracy. The DA and phasor-FLIM method may provide the opportunity to improve implantation success rates for in vitro fertilization clinics.

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere.

Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD + ) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer's disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by 89% to levels corresponding to the young neurons. Using FLIM as a non-invasive approach, we were able to measure the reversibility of aging subcellular free NADH levels in live neurons. Our results suggest a potential reductive treatment to reverse the loss of free NADH in old and Alzheimer's neurons.

The angiotensin II AT 1 and the endothelin 1 ET A receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates. Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required for Fluorescence Resonance Energy Transfer measurements. Our results showed that stably transfected angiotensin II AT 1 receptors and transiently transfected endothelin 1 ET A receptors, were found as monomeric, dimeric, and tetrameric receptor aggregates. Interestingly, the binding of antihypertensive agents like losartan and BQ123, earlier suggested to be inverse agonists, significantly increased the proportion of monomers and reduced the occurrence of dimers on the cell membrane; while the kown endothelin 1 ET A antagonist sitaxentan did not influence the aggregation state of these receptors., (Copyright © 2019. Published by Elsevier B.V.)

Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.

Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish individual cells based on their metabolic states, which could be used as an upstream phenotypic platform to correlate with genomic statistics. © 2018 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

Extracellular matrix (ECM) mechanical properties upregulate cancer invasion, cell contractility, and focal adhesion formation. Alteration in energy metabolism is a known characteristic of cancer cells (i.e., Warburg effect) and modulates cell invasion. There is little evidence to show if collagen density can alter cancer cell metabolism. We investigated changes in energy metabolism due to collagen density in five breast cell lines by measuring the fluorescence lifetime of NADH. We found that only triple-negative breast cancer cells, MDA-MB231 and MDA-MB468 cells, had an increased population of bound NADH, indicating an oxidative phosphorylation (OXPHOS) signature, as collagen density decreased. When inhibiting ROCK and cell contractility, MDA-MB231 cells on glass shifted from glycolysis (GLY) to OXPHOS, confirming the intricate relationship between mechanosensing and metabolism. MCF10A cells showed less significant changes in metabolism, shifting towards GLY as collagen density decreased. The MCF-7 and T-47D, less invasive breast cancer cells, compared to the MDA-MB231 and MDA-MB468 cells, showed no changes regardless of substrate. In addition, OXPHOS or GLY inhibitors in MDA-MB231 cells showed dramatic shifts from OXPHOS to GLY or vice versa. These results provide an important link between cellular metabolism, contractility, and collagen density in human breast cancer.

We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation, revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores, suggesting that macropinocytosis can supply nutrients other than amino acids. Nontransformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically. Significance: As PTEN-deficient prostate cancer cells proliferate in low-nutrient environments by scavenging necrotic debris and extracellular protein via macropinocytosis, blocking macropinocytosis by inhibiting AMPK, RAC1, or PI3K may have therapeutic value, particularly in necrotic tumors and in combination with therapies that cause nutrient stress. Cancer Discov; 8(7); 866-83. ©2018 AACR. See related commentary by Commisso and Debnath, p. 800 This article is highlighted in the In This Issue feature, p. 781 ., (©2018 American Association for Cancer Research.)

Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals' neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections., ((2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).)

The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence screening. The trapped single leukemia cells, e.g., THP-1, Jurkat and K562 cells, are distinguished from WBCs in the phasor-FLIM lifetime map, as they exhibit significant shift towards shorter fluorescence lifetime and a higher ratio of free/bound NADH compared to WBCs, because of their glycolysis-dominant metabolism for rapid proliferation. Based on a multiparametric scheme comparing the eight parameter-spectra of the phasor-FLIM signatures, spiked leukemia cells are quantitatively distinguished from normal WBCs with an area-under-the-curve (AUC) value of 1.00. Different leukemia cell lines are also quantitatively distinguished from each other with AUC values higher than 0.95, demonstrating high sensitivity and specificity for single cell analysis. The presented platform is the first to enable high-density size-based single-cell trapping simultaneously with RBC filtering and rapid label-free individual-leukemia-cell screening through non-invasive metabolic imaging. Compared to conventional biomolecular diagnostics techniques, phasor-FLIM based single-cell screening is label-free, cell-friendly, robust, and has the potential to screen blood in clinical volumes through parallelization.

Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lung cancer is one of the deadliest cancers in the world because of chemo-resistance to the commonly used cisplatin-based treatments. The use of low fidelity DNA polymerases in the translesional synthesis (TLS) DNA damage response pathway that repairs lesions caused by cisplatin also presents a mutational carcinogenic burden on cells that needs to be regulated by the tumor suppressor protein p53. However, there is much debate over the roles of the reversionless 3-like (REV3L) protein responsible for TLS and p53 in regulating cancer cell metabolism. In this study, the fluorescence lifetime of the metabolic coenzyme NADH reveals that the absence of REV3L can promote the p53-mediated upregulation of oxidative phosphorylation in cisplatin-treated H1299 lung carcinoma cells and increases cancer cell sensitivity to this platinum-based chemotherapy. These results demonstrate a previously unrecognized relationship between p53 and REV3L in cancer cell metabolism and may lead to improvements in chemotherapy treatment plans that reduce cisplatin resistance in lung cancer., (Copyright © 2018. Published by Elsevier Inc.)

Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.

Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.

Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive correlation between free-NADH fraction and cell growth, which was related to an increase of TMC fraction. In comparison, the increase of EGFR and cellular respirations preceded all these changes. In conclusion, FLIM of NADH provides a non-invasive method to monitor the dynamics of tumor heterogeneity before and after treatment., Competing Interests: The authors declare that they have no personal circumstances or interest that may be perceived as inappropriately influencing the representation or interpretation of reported research results.

Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter-driven mesenchymal stem cell (MSC)-based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer-associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to their long persistence in the body (measured in years), making them refractory to the development of resistance to treatment. Our MRCS can serve as a platform for future diagnostics and therapies targeting aberrant tissue stiffness in conditions such as cancer and fibrotic diseases, and it should help to elucidate mechanobiology and reveal what cells "feel" in the microenvironment in vivo., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle are only partially understood. Here we analyze the spatial distribution of Cdc42 activity by moni-toring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion decreased Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi but were associated with a substantial reduction in PM-associated Cdc42 activity. Of interest, cells with reduced Cdc42 activity at the PM displayed altered centrosome morphology, suggesting that centrosome regulation may be mediated by active Cdc42 at the PM. Our study describes a novel quantitative approach to determine Cdc42 activity at specific subcellular locations and reveals new regulatory principles and functions of this small GTPase., (© 2017 Herrington et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Selective cell adhesion is desirable to control cell growth and migration on biomedical implants. Mesenchymal cell migration is regulated through focal adhesions (FAs) and can be modulated by their microenvironment, including changes in surface topography. We use the Number and Molecular Brightness (N&B) imaging analysis to provide a unique perspective on FA assembly and disassembly. This imaging analysis generates a map of real-time fluctuations of protein monomers, dimers, and higher order aggregates of FA proteins, such as paxillin during assembly and disassembly. We show a dynamic view of how nanostructured surfaces (nanoline gratings or nanopillars) regulate single molecular dynamics. In particular, we report that the smallest nanopillars (100 nm spacing) gave rise to a low population of disassembling adhesion clusters of ∼2 paxillin proteins whereas the larger nanopillars (380 nm spacing) gave rise to a much larger population of larger disassembling clusters of ∼3-5 paxillin proteins. Cells were more motile on the smaller nanopillars (spaced 100-130 nm apart) compared to all other surfaces studied. Thus, physical nanotopography influences cell motility, adhesion size, and adhesion assembly and disassembly. We report for the first time, with single molecular detection, how nanotopography influences cell motility and protein reorganization in adhesions.

Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by-products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distribution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that increasing SIRT1 expression in oocyte nuclei was inversely related to decreasing free/bound NADH during the primordial to primary follicle transition. We characterized oocyte metabolism at these early stages to be NADH producing (glycolysis/Krebs). We extended the results of prior studies to show that cumulus and mural granulosa cell metabolism in secondary through preovulatory follicles is mainly NADH producing (glycolysis/Krebs cycle), while oocyte metabolism is mainly NADH consuming (oxidative phosphorylation). Taken together, our data characterize dynamic changes in free/bound NADH and SIRT1 expression during early follicular development and confirm results from previous studies defining antral and preovulatory follicle metabolism in culture., (© 2016 by the Society for the Study of Reproduction, Inc.)

The delivery of therapeutics to the central nervous system (CNS) remains a major challenge in part due to the presence of the blood-brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo . Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro . This study encourages further development of engineered exosomes as drug delivery vehicles or tracking tools for treating or monitoring neurological diseases.

Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher free NADH suggesting a possible synergism between metabolic dysfunction and transcriptional regulation. Our results indicate that metabolic dysfunction in HD shifts to increased glycolysis leading to oxidative stress and cell death. This powerful label free method can be used to screen native HD tissue samples and for potential drug screening.

Single pores in the resistive-pulse technique are used as an analytics tool to detect, size, and characterize physical as well as chemical properties of individual objects such as molecules and particles. Each object passing through a pore causes a transient change of the transmembrane current called a resistive pulse. In high salt concentrations when the pore diameter is significantly larger than the screening Debye length, it is assumed that the particle size and surface charge can be determined independently from the same experiment. In this article we challenge this assumption and show that highly charged hard spheres can cause a significant increase of the resistive-pulse amplitude compared to neutral particles of a similar diameter. As a result, resistive pulses overestimate the size of charged particles by even 20%. The observation is explained by the effect of concentration polarization created across particles in a pore, revealed by numerical modeling of ionic concentrations, ion current, and local electric fields. It is notable that in resistive-pulse experiments with cylindrical pores, concentration polarization was previously shown to influence ionic concentrations only at pore entrances; consequently, additional and transient modulation of resistive pulses was observed when a particle entered or left the pore. Here we postulate that concentration polarization can occur across transported particles at any particle position along the pore axis and affect the magnitude of the entire resistive pulse. Consequently, the recorded resistive pulses of highly charged particles reflect not only the particles' volume but also the size of the depletion zone created in front of the moving particle. Moreover, the modeling identified that the effective surface charge density of particles depended not only on the density of functional groups on the particle but also on the capacitance of the Stern layer. The findings are of crucial importance for sizing particles and characterizing their surface charge properties.

Unlabelled: Fluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system's symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed flow motions, when compared to SpatioTemporal Image Correlation Spectroscopy (STICS). Although we present a specific example (lipoplexes in living cells), the emphasis is in the discussion of the method, its basic assumptions and its validation on numeric simulations., Statement of Significance: Recent advances in nanoparticle-based drug and gene delivery systems have pointed out the interactions at cellular and subcellular levels as key-factors for the efficiency of the adopted biomaterials. Such biochemical and biophysical interactions drive and affect the intracellular dynamics, that is commonly characterized by means of fluorescence microscopy and spectroscopy techniques. Here we present a novel Image Correlation Spectroscopy (ICS) method as a promising tool to capture the intracellular behavior of nanoparticles with high resolution and low background's sensitivity. This study overcomes some of the approximations adopted so far, by decoupling the flow terms of the investigated dynamics and thus recovering ensemble's information from specific single particle behaviors. Finally, relevant implications for nanoparticle-based drug delivery are shown., (Copyright © 2016 Acta Materialia Inc. All rights reserved.)

We report a self-assisted optothermal trapping and patterning of gold nanorods (GNRs) on glass surfaces with a femtosecond laser. We show that GNRs are not only the trapping targets, but also can enhance the optothermal trapping of other particles. This trapping phenomenon is the net result of thermophoresis and a convective flow caused by localized heating. The heating is due to the conversion of absorbed photons into heat at GNR's longitudinal surface plasmon resonance (LSPR) wavelength. First, we investigated the optothermal trapping of GNRs at their LSPR wavelength on the glass surface with as low as 0.5 mW laser power. The trapping range was observed to be larger than a typical field of view, e.g. 210 µm  ×  210 µm here. Second, by adjusting the distance between the laser focus and the glass surface, ring patterns of GNRs on the glass surface were obtained. These patterns could be controlled by the laser power and the numerical aperture of the microscope objective. Moreover, we examined the spectral emission of GNRs under different trapping conditions using the spectral phasor approach to reveal the temperature and association status of GNRs. Our study will help understanding manipulation of flows in solution and in biological systems that can be applied in future investigations of GNR-induced heating and flows.

We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution.

Quantification of miRNAs in blood can be potentially used for early disease detection, surveillance monitoring and drug response evaluation. However, quantitative and robust measurement of miRNAs in blood is still a major challenge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays. Here, we present the 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) system where the plasma sample containing target miRNAs is encapsulated into microdroplets, enzymatically amplified and digitally counted using a novel, high-throughput 3D particle counter. Using Let-7a as a target, we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10s to 10 000 copies per mL in ≤3 hours without the need for sample processing such as RNA extraction. Using this new tool, we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that in healthy donor samples. Our IC 3D system has the potential to introduce a new paradigm for rapid, sensitive and specific detection of low-abundance biomarkers in biological samples with minimal sample processing.

The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell.