Single-cell Imaging Research Articles

The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal

Senescence, a state of irreversible cell-cycle withdrawal, is difficult to distinguish from quiescence, a state of reversible cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether these states are truly distinct. To address this, we use single-cell time-lapse imaging to distinguish slow-cycling cells that spend long periods in quiescence from cells that never cycle after recovery from senescence-inducing treatments, followed by staining for various senescence biomarkers. We find that the staining intensity of multiple senescence biomarkers is graded rather than binary and reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data show that quiescent and apparent senescent cells are nearly molecularly indistinguishable from each other at a snapshot in time. This suggests that cell-cycle withdrawal itself is graded rather than binary, where the intensities of senescence biomarkers integrate the duration of past cell-cycle withdrawal.

Single-cell multimodal imaging uncovers energy conversion pathways in biohybrids.

Microbe-semiconductor biohybrids, which integrate microbial enzymatic synthesis with the light-harvesting capabilities of inorganic semiconductors, have emerged as promising solar-to-chemical conversion systems. Improving the electron transport at the nano-bio interface and inside cells is important for boosting conversion efficiencies, yet the underlying mechanism is challenging to study by bulk measurements owing to the heterogeneities of both constituents. Here we develop a generalizable, quantitative multimodal microscopy platform that combines multi-channel optical imaging and photocurrent mapping to probe such biohybrids down to single- to sub-cell/particle levels. We uncover and differentiate the critical roles of different hydrogenases in the lithoautotrophic bacterium Ralstonia eutropha for bioplastic formation, discover this bacterium's surprisingly large nanoampere-level electron-uptake capability, and dissect the cross-membrane electron-transport pathways. This imaging platform, and the associated analytical framework, can uncover electron-transport mechanisms in various types of biohybrid, and potentially offers a means to use and engineer R. eutropha for efficient chemical production coupled with photocatalytic materials.

Optical single-pixel volumetric imaging by three-dimensional light-field illumination

Three-dimensional single-pixel imaging (3D SPI) has become an attractive imaging modality for both biomedical research and optical sensing. 3D-SPI techniques generally depend on time-of-flight or stereovision principle to extract depth information from backscattered light. However, existing implementations for these two optical schemes are limited to surface mapping of 3D objects at depth resolutions, at best, at the millimeter level. Here, we report 3D light-field illumination single-pixel microscopy (3D-LFI-SPM) that enables volumetric imaging of microscopic objects with a near-diffraction-limit 3D optical resolution. Aimed at 3D space reconstruction, 3D-LFI-SPM optically samples the 3D Fourier spectrum by combining 3D structured light-field illumination with single-element intensity detection. We build a 3D-LFI-SPM prototype that provides an imaging volume of ∼390 × 390 × 3,800 μm3 and achieves 2.7-μm lateral resolution and better than 37-μm axial resolution. Its capability of 3D visualization of label-free optical absorption contrast is demonstrated by imaging single algal cells in vivo. Our approach opens broad perspectives for 3D SPI with potential applications in various fields, such as biomedical functional imaging.

L-Carnitine Suppresses Transient Receptor Potential Vanilloid Type 1 Activation in Human Corneal Epithelial Cells.

Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca2+ influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T). Single-cell fluorescence imaging of calcein/AM-loaded cells or fura-2/AM-labeled cells was used to evaluate cell volume changes and intracellular calcium levels, respectively. Planar patch-clamp technique was used to measure whole-cell currents. TRPV1 activation via either capsaicin (20 µmol/L), hyperosmolarity (≈450 mosmol/L) or an increase in ambient bath temperature to 43 °C induced intracellular calcium transients and augmented whole-cell currents, whereas hypertonicity induced cell volume shrinkage. In contrast, either capsazepine (10 µmol/L) or L-carnitine (1-3 mmol/L) reduced all these responses. Taken together, L-carnitine and capsazepine suppress hypertonicity-induced TRPV1 activation by blocking cell volume shrinkage.

Copper chelation suppresses epithelial-mesenchymal transition by inhibition of canonical and non-canonical TGF-β signaling pathways in cancer

BackgroundMetastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis.ResultsOur cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment.ConclusionsOur study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a diverse range of cancers.

Label-free mid-infrared photothermal live-cell imaging beyond video rate

Advancement in mid-infrared (MIR) technology has led to promising biomedical applications of MIR spectroscopy, such as liquid biopsy or breath diagnosis. On the contrary, MIR microscopy has been rarely used for live biological samples in an aqueous environment due to the lack of spatial resolution and the large water absorption background. Recently, mid-infrared photothermal (MIP) imaging has proven to be applicable to 2D and 3D single-cell imaging with high spatial resolution inherited from visible light. However, the maximum measurement rate has been limited to several frames s−1, limiting its range of use. Here, we develop a significantly improved wide-field MIP quantitative phase microscope with two orders-of-magnitude higher signal-to-noise ratio than previous MIP imaging techniques and demonstrate live-cell imaging beyond video rate. We first derive optimal system design by numerically simulating thermal conduction following the photothermal effect. Then, we develop the designed system with a homemade nanosecond MIR optical parametric oscillator and a high full-well-capacity image sensor. Our high-speed and high-spatial-resolution MIR microscope has great potential to become a new tool for life science, in particular for live-cell analysis.

Maternal age, obesity and hyperglycaemia are associated with a delay in preimplantation embryo development in mouse.

Fertility has decreased due to advanced maternal age and the rising prevalence of the metabolic syndrome. Using quantitative image analysis methods, we show that these factors are associated with delayed preimplantation embryo development in a mouse model. Delayed maternal age, obesity and diabetes are associated with reduced fertility. We investigated how age and obesity/metabolic syndrome impact fertility and hypothesized that its decrease is due to defects in preimplantation embryo development. Three groups of female C57Bl6 mice (12 weeks, 9 months and 1 year old) were fed either a high-fat diet for 8 weeks, to induce obesity and the metabolic syndrome, or a control chow diet. Body weight and composition, glucose tolerance and insulin resistance were assessed. Fecundity was evaluated by mating and pregnancy rates, as well as by the number of embryos. Embryo quality was assessed morphologically, and cell fate composition was analysed in preimplantation embryos by state-of-the-art single-cell quantitative confocal image analysis. The high-fat diet was associated with increased adiposity, glucose intolerance and insulin resistance, especially in the older mice. Fecundity was affected by age more than by the diet. Both age and high-fat diet were associated with reduced cell fate allocation, indicating a delay in the preimplantation embryo development, and with increased expression of GATA3, an inhibitor of placentation. These results support that age and the metabolic syndrome reduce fertility through mechanisms which are present at conception or very early in pregnancy.

Single-cell imaging and transcriptomic analyses of firm adhesion between patient-derived cancer and endothelial cells under shear stress

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is a key event during tumour progression and metastasis formation. However, the analysis of its underlying cellular and molecular mechanisms is largely limited by the intrinsic difficulties to study the interactions between circulating cancer cells and endothelial cells in vivo, and in vitro under conditions that mimic the in vivo blood flow. Here, we developed a method to study cell:cell firm adhesion under shear-stress conditions coupled to high-content live-cell imaging, and single-cell RNAseq analysis. As the model system, we used cancer cells freshly isolated from patient-derived xenografts (PDXs) and human primary endothelial cells. Breast cancer is the most common cancer in women worldwide and the leading cause of cancer-related deaths among women. Therefore, we set up protocols for breast cancer PDX tumour dissociation, isolation and purification to obtain freshly isolated PDX-derived human cancer single cell suspension. We then implemented an in vitro assay to study cancer to endothelial cells firm adhesion under shear-stress, using an all–human microfluidic model coupled to time-lapse and live-cell imaging. Finally, we developed a method to successfully retrieve, separate and enrich alive endothelial and cancer cells from the flow-based firm adhesion assay. Most notably, we used retrieved cells for single-cell RNAseq analysis and showed that samples quality, number of cells and transcripts per cell were consistent and optimal for downstream discovery analyses. In conclusion, we developed a workflow method that can provide insights into the mechanisms of cancer adhesion to endothelial cells, and identify new targets for personalized treatments development for the clinic to prevent and/or treat breast cancer metastasis formation.

Bioluminescent Genetically Encoded Glutamate Indicators for Molecular Imaging of Neuronal Activity.

Genetically encoded optical sensors and advancements in microscopy instrumentation and techniques have revolutionized the scientific toolbox available for probing complex biological processes such as release of specific neurotransmitters. Most genetically encoded optical sensors currently used are based on fluorescence and have been highly successful tools for single-cell imaging in superficial brain regions. However, there remains a need to develop new tools for reporting neuronal activity in vivo within deeper structures without the need for hardware such as lenses or fibers to be implanted within the brain. Our approach to this problem is to replace the fluorescent elements of the existing biosensors with bioluminescent elements. This eliminates the need of external light sources to illuminate the sensor, thus allowing deeper brain regions to be imaged noninvasively. Here, we report the development of the first genetically encoded neurotransmitter indicators based on bioluminescent light emission. These probes were optimized by high-throughput screening of linker libraries. The selected probes exhibit robust changes in light output in response to the extracellular presence of the excitatory neurotransmitter glutamate. We expect this new approach to neurotransmitter indicator design to enable the engineering of specific bioluminescent probes for multiple additional neurotransmitters in the future, ultimately allowing neuroscientists to monitor activity associated with a specific neurotransmitter as it relates to behavior in a variety of neuronal and psychiatric disorders, among many other applications.

Aerosolized miR-138-5p and miR-200c targets PD-L1 for lung cancer prevention.

The development of chemopreventive strategies with the ability to prevent the progression of lung lesions to malignant cancers would reduce the mortality and morbidity resulting from this deadly disease. Delivery of microRNA (miRNA) by inhalation is a novel method for lung cancer prevention. In this study, we investigated the combined efficacy of aerosolized miR-138-5p and miR-200c miRNA mimics in lung cancer prevention. Combination of the two miRNAs inhibited Benzo(a)pyrene (B((a))P)-induced lung adenomas and N-nitroso-tris-chloroethylurea (NTCU)-induced lung squamous cell carcinomas with no detectable side effects. Using single-cell RNA sequencing (scRNA-seq) and imaging mass cytometry (IMC), we found that both miRNAs inhibited programmed cell death ligand 1 (PD-L1) expression. Our flow cytometry results showed that aerosolized delivery of combined miRNAs increased CD4+ and CD8+ T cells and reduced the expression of programmed cell death protein 1 (PD-1) and T-regulatory cells. Our results demonstrated that the delivery of aerosolized microRNAs targeting PD-L1 can be highly effective in preventing lung cancer development and progression in mice.

Detection of antimicrobial impact on gram-negative bacterial cell envelope based on single-cell imaging by scanning electron microscopy

Rapid determination of drug efficacy against bacterial pathogens is needed to detect potentially resistant bacteria and allow for more rational use of antimicrobials. As an indicator of the antimicrobial effect for rapid detection, we found changes in image brightness in antimicrobial-affected bacteria by scanning electron microscopy (SEM). The cell envelopes of unaffected bacteria were stained with phosphotungstic acid (PTA), whereas the entire cells of affected bacteria were stained. Since tungsten density increases backscattered electron intensity, brighter bacterial images indicate lethal damage. We propose a simplified method for determining antimicrobial efficacy by detecting damage that occurs immediately after drug administration using tabletop SEM. This method enabled the visualization of microscopic deformations while distinguishing bacterial-cell-envelope damage on gram-negative bacteria due to image-brightness change. Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa were exposed to imipenem and colistin, which affect the cell envelope through different mechanisms. Classification of single-cell images based on brightness was quantified for approximately 500 bacteria per sample, and the bright images predominated within 5 to 60 min of antimicrobial treatment, depending on the species. Using intracellular PTA staining and characteristic deformations as indicators, it was possible to determine the efficacy of antimicrobials in causing bacterial-cell-envelope damage.

Repeatability and reproducibility of apparent exchange rate measurements in yeast cell phantoms using filter-exchange imaging

ObjectivesDevelopment of a protocol for validation and quality assurance of filter-exchange imaging (FEXI) pulse sequences with well-defined and reproducible phantoms.Materials and methodsA FEXI pulse sequence was implemented on a 7 T preclinical MRI scanner. Six experiments in three different test categories were established for sequence validation, demonstration of the reproducibility of phantoms and the measurement of induced changes in the apparent exchange rate (AXR). First, an ice–water phantom was used to investigate the consistency of apparent diffusion coefficient (ADC) measurements with different diffusion filters. Second, yeast cell phantoms were utilized to validate the determination of the AXR in terms of repeatability (same phantom and session), reproducibility (separate but comparable phantoms in different sessions) and directionality of diffusion encodings. Third, the yeast cell phantoms were, furthermore, used to assess potential AXR bias because of altered cell density and temperature. In addition, a treatment experiment with aquaporin inhibitors was performed to evaluate the influence of these compounds on the cell membrane permeability in yeast cells.ResultsFEXI-based ADC measurements of an ice–water phantom were performed for three different filter strengths, showed good agreement with the literature value of 1.099 × 10–3 mm2/s and had a maximum coefficient of variation (CV) of 0.55% within the individual filter strengths. AXR estimation in a single yeast cell phantom and imaging session with five repetitions resulted in an overall mean value of (1.49 ± 0.05) s−1 and a CV of 3.4% between the chosen regions of interest. For three separately prepared phantoms, AXR measurements resulted in a mean value of (1.50 ± 0.04) s−1 and a CV of 2.7% across the three phantoms, demonstrating high reproducibility. Across three orthogonal diffusion directions, a mean value of (1.57 ± 0.03) s−1 with a CV of 1.9% was detected, consistent with isotropy of AXR in yeast cells. Temperature and AXR were linearly correlated (R2 = 0.99) and an activation energy EA of 37.7 kJ/mol was determined by Arrhenius plot. Furthermore, a negative correlation was found between cell density (as determined by the reference ADC/fe) and AXR (R2 = 0.95). The treatment experiment resulted in significantly decreased AXR values at different temperatures in the treated sample compared to the untreated control indicating an inhibiting effect.ConclusionsUsing ice–water and yeast cell-based phantoms, a protocol for the validation of FEXI pulse sequences was established for the assessment of stability, repeatability, reproducibility and directionality. In addition, a strong dependence of AXR on cell density and temperature was shown. As AXR is an emerging novel imaging biomarker, the suggested protocol will be useful for quality assurance of AXR measurements within a study and potentially across multiple sites.

A Single-Cell Atlas of Small Airway Disease in Chronic Obstructive Pulmonary Disease: A Cross-Sectional Study.

Rationale: Emerging data demonstrate that the smallest conducting airways, terminal bronchioles, are the early site of tissue destruction in chronic obstructive pulmonary disease (COPD) and are reduced by as much as 41% by the time someone is diagnosed with mild (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 1) COPD. Objectives: To develop a single-cell atlas that describes the structural, cellular, and extracellular matrix alterations underlying terminal bronchiole loss in COPD. Methods: This cross-sectional study of 262 lung samples derived from 34 ex-smokers with normal lung function (n = 10) or GOLD stage 1 (n = 10), stage 2 (n = 8), or stage 4 (n = 6) COPD was performed to assess the morphology, extracellular matrix, single-cell atlas, and genes associated with terminal bronchiole reduction using stereology, micro-computed tomography, nonlinear optical microscopy, imaging mass spectrometry, and transcriptomics. Measurements and Main Results: The lumen area of terminal bronchioles progressively narrows with COPD severity as a result of the loss of elastin fibers within alveolar attachments, which was observed before microscopic emphysematous tissue destruction in GOLD stage 1 and 2 COPD. The single-cell atlas of terminal bronchioles in COPD demonstrated M1-like macrophages and neutrophils located within alveolar attachments and associated with the pathobiology of elastin fiber loss, whereas adaptive immune cells (naive, CD4, and CD8 T cells, and B cells) are associated with terminal bronchiole wall remodeling. Terminal bronchiole pathology was associated with the upregulation of genes involved in innate and adaptive immune responses, the interferon response, and the degranulation of neutrophils. Conclusions: This comprehensive single-cell atlas highlights terminal bronchiole alveolar attachments as the initial site of tissue destruction in centrilobular emphysema and an attractive target for disease modification.

Sensitivity of endogenous autofluorescence in HeLa cells to the application of external magnetic fields

Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at the cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. It has been proposed that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we investigate the magnetic field sensitivity of an endogenous autofluorescence in HeLa cells. Under the experimental conditions used, magnetic field sensitivity of an endogenous autofluorescence was not observed in HeLa cells. We present a number of arguments indicating why this is the case in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level.

Cyclin E/CDK2 and feedback from soluble histone protein regulate the S phase burst of histone biosynthesis

Faithful DNA replication requires that cells fine-tune their histone pool in coordination with cell-cycle progression. Replication-dependent histone biosynthesis is initiated at a low level upon cell-cycle commitment, followed by a burst at the G1/S transition, but it remains unclear how exactly the cell regulates this burst in histone biosynthesis as DNA replication begins. Here, we use single-cell time-lapse imaging to elucidate the mechanisms by which cells modulate histone production during different phases of the cell cycle. We find that CDK2-mediated phosphorylation of NPAT at the restriction point triggers histone transcription, which results in a burst of histone mRNA precisely at the G1/S phase boundary. Excess soluble histone protein further modulates histone abundance by promoting the degradation of histone mRNA for the duration of S phase. Thus, cells regulate their histone production in strict coordination with cell-cycle progression by two distinct mechanisms acting in concert.

Precisely timed regulation of enhancer activity defines the binary expression pattern of Fushi tarazu in the Drosophila embryo.

The genes that drive development each typically have many different enhancers. Properly coordinating the action of these different enhancers is crucial to correctly specifying cell-fate decisions, yet it remains poorly understood how their activity is choregraphed in time. To shed light on this question, we used recently developed single-cell live imaging tools to dissect the regulation of Fushi tarazu (Ftz) in Drosophila melanogaster embryos. Ftz is a transcription factor that is expressed in asymmetric stripes by two distinct enhancers: autoregulatory and zebra. The anterior edge of each stripe needs to be sharply defined to specify essential lineage boundaries. Here, we tracked how boundary cells commit to either a high-Ftz or low-Ftz fate by measuring Ftz protein traces in real time and simultaneously quantifying transcription from the endogenous locus and individual enhancers. This revealed that the autoregulatory enhancer does not establish this fate choice. Instead, it perpetuates the decision defined by zebra. This is contrary to the prevailing view that autoregulation drives the fate decision by causing bi-stable Ftz expression. Furthermore, we showed that the autoregulatory enhancer is not activated based on a Ftz-concentration threshold but through a timing-based mechanism. We hypothesize that this is regulated by several ubiquitously expressed pioneer-like transcription factors, which have recently been shown to act as timers in the embryo. Our work provides new insight into how precisely timed enhancer activity can directly regulate the dynamics of gene regulatory networks, which may be a general mechanism for ensuring that embryogenesis runs like clockwork.

Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development.

Automated imaging techniques have been in increasing demand for the more advanced analysis and efficient characterization of cellular phenotypes. The success of the image-based profiling method hinges on assays that can rapidly and simultaneously capture a wide range of phenotypic features. We have developed an automated image acquisition method for fungal cytological profiling (FCP) using an imaging flow cytometer that can objectively measure over 250 features of a single fungal cell. Fungal cells were labeled with calcofluor white and FM4-64FX, which bind to the cell wall and lipophilic membrane, respectively. Images of single cells were analyzed using IDEAS® software. We first acquired FCPs of fungal cells treated with fluconazole, amphotericin B, and caspofungin, each with a distinct mode of action, to establish FCP databases of profiles associated with specific antifungal treatment. Once fully established, we investigated the potential application of this technique as a screening methodology to identify compounds with novel antifungal activity against Candida albicans and Cryptococcus neoformans. Altogether, we have developed a rapid, powerful, and novel image-profiling method for the phenotypic characterization of fungal cells, also with potential applications in antifungal drug development.

An automated malaria cells detection from thin blood smear images using deep learning.

Timely and rapid diagnosis is crucial for faster and proper malaria treatment planning. Microscopic examination is the gold standard for malaria diagnosis, where hundreds of millions of blood films are examined annually. However, this method's effectiveness depends on the trained microscopist's skills. With the increasing interest in applying deep learning in malaria diagnosis, this study aims to determine the most suitable deep-learning object detection architecture and their applicability to detect and distinguish red blood cells as either malaria-infected or non-infected cells. The object detectors Yolov4, Faster R-CNN, and SSD 300 are trained with images infected by all five malaria parasites and from four stages of infection with 80/20 train and test data partition. The performance of object detectors is evaluated, and hyperparameters are optimized to select the best-performing model. The best-performing model was also assessed with an independent dataset to verify the models' ability to generalize in different domains. The results show that upon training, the Yolov4 model achieves a precision of 83%, recall of 95%, F1-score of 89%, and mean average precision of 93.87% at a threshold of 0.5. Conclusively, Yolov4 can act as an alternative in detecting the infected cells from whole thin blood smear images. Object detectors can complement a deep learning classification model in detecting infected cells since they eliminate the need to train on single-cell images and have been demonstrated to be more feasible for a different target domain.

A novel melanocortin fusion protein inhibits fibrinogen oxidation and degradation during trauma-induced coagulopathy

AbstractImmune cell inflammation is implicated in the pathophysiology of acute trauma-induced coagulopathy (TIC). We hypothesized that leukocyte inflammation contributes to TIC through the oxidation and proteolysis of fibrinogen. To test this hypothesis, antioxidants and a novel anti-inflammatory melanocortin fusion protein (AQB-565) were used to study the effects of interleukin-6 (IL-6)–stimulated human leukocytes on fibrinogen using single-cell imaging flow cytometry and multiplex fluorescent western blotting. We also studied the effects of AQB-565 on fibrinogen using an in vivo rat trauma model of native TIC. IL-6 induced cellular inflammation and mitochondrial superoxide production in human monocytes, causing fibrinogen oxidation and degradation in vitro. Antioxidants suppressing mitochondrial superoxide reduced oxidative stress and inflammation and protected fibrinogen. AQB-565 decreased inflammation, inhibited mitochondrial superoxide, and protected fibrinogen in vitro. Trauma with hemorrhagic shock increased IL-6 and other proinflammatory cytokines and chemokines, selectively oxidized and degraded fibrinogen, and induced TIC in rats in vivo. AQB-565, given at the onset of hemorrhage, blocked inflammation, protected fibrinogen from oxidation and degradation, and prevented TIC. Leukocyte activation contributes to TIC through the oxidation and degradation of fibrinogen, which involves mitochondrial superoxide and cellular inflammation. Suppression of inflammation by activation of melanocortin pathways may be a novel approach for the prevention and treatment of TIC.

Synthesis, Characterization, and Applications of a Superior Dendrimer-Based Polymer for Multiplexed Ion Beam Imaging Time-of-Flight Analysis.

High-dimensional single-cell mass spectrometric imaging techniques such as multiplexed ion beam imaging by time-of-flight mass spectrometry (MIBI-TOF), imaging mass cytometry (IMC), and flow cytometry-based CyTOF utilize antibodies conjugated to linear metal-chelating polymers. Here, we report on the synthesis and characterization of a dendrimer-based polymer and its utilization in tissue imaging using MIBI-TOF. We compared the staining performance in FFPE tissue of antibodies for lineage-specific immune proteins (CD20, CD3, CD45, FoxP3) that were conjugated with dendrimer or linear polymer. Staining of serial tissue sections with dendron-conjugated and linear-polymer-conjugated antibodies revealed comparable avidities of dendrons and linear polymers with log2 (ratio of mean positive pixel intensity of staining for linear polymers to dendrons) within the range ±0.25. Interestingly, dendron-conjugated antibodies were observed to have some advantages over linear polymer-conjugated antibodies. For example, tissue staining of a nuclear protein, FoxP3 with dendron-conjugated antibodies showed notably less background staining than that of linear-polymer-conjugated antibodies. Additionally, dendron-conjugated antibodies did not exhibit off-target cytosolic binding in neural tissue typically observed when using linear polymer conjugates. Taken together, this work provides a versatile framework for using third-generation dendron-conjugated antibodies with improved staining over conventional linear polymers.