Ebola virus-like particles reprogram cellular metabolism.

Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. KEY MESSAGES: • Ebola VLP can lead to metabolic alternations in endothelial cells and M1 and M2 macrophages. • Differential abundance of metabolites, mainly including fatty acids and sterol lipids, was observed after Ebola VLP exposure. • Multiple fatty acid-, steroid-, and amino acid-related metabolism pathways were observed perturbed.

Capillary microsampling-based single-cell metabolomics by mass spectrometry and its applications in medicine and drug discovery.

Characterization of cellular metabolic states is a technical challenge in biomedicine. Cellular heterogeneity caused by inherent diversity in expression of metabolic enzymes or due to sensitivity of metabolic reactions to perturbations, necessitates single cell analysis of metabolism. Heterogeneity is typically seen in cancer and thus, single-cell metabolomics is expectedly useful in studying cancer progression, metastasis, and variations in cancer drug response. However, low sample volumes and analyte concentrations limit detection of critically important metabolites. Capillary microsampling-based mass spectrometry approaches are emerging as a promising solution for achieving single-cell omics. Herein, we focus on the recent advances in capillary microsampling-based mass spectrometry techniques for single-cell metabolomics. We discuss recent technical developments and applications to cancer medicine and drug discovery.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

Direct infusion nano-electrospray ionization mass spectrometry for therapeutic drug monitoring of ciprofloxacin and its metabolites in human saliva.

A rapid and sensitive method based on direct infusion-nano-electrospray ionization mass spectrometry (DI-nESI-MS) has been developed for the detection and quantification of ciprofloxacin and its metabolites in human saliva. Saliva samples were collected after the oral administration of 500 mg ciprofloxacin tablets. Internal standard (IS), tamoxifen, was added to the collected samples, and then diluted with the ionization solvent, centrifuged and filtered. An aliquot of 4 µL of the filtrate was loaded into a nanospray (NS) capillary. The NS capillary was then fitted into an off-line ion source and the instrument was operated to acquire a two-minute run by applying a voltage of 1000 V (positive-ion detection mode). Quantification of ciprofloxacin relied on the ratio of its peak intensity to the IS peak intensity. The DI-nESI-MS method was validated and provided satisfactory precision with relative standard deviation ranging from 0.39 to 7.48 % and accuracy with relative error ranging from -2.12 to 9.72 %. The calibration curve showed good linearity (r2) > 0.999 over the concentration range of 10-4000 ng/mL. These results verify the effectiveness of the DI-nESI-MS method for monitoring of ciprofloxacin and its metabolites in human saliva samples.

Lassa hemorrhagic shock syndrome-on-a-chip.

Lack of experimental human models hinders research on Lassa hemorrhagic fever and the development of treatment strategies. Here, we report the first chip-based model for Lassa hemorrhagic syndrome. The chip features a microvessel interfacing collagen network as a simple mimic for extracellular matrix, allowing for quantitative and real-time vascular integrity assessment. Luminal infusion of Lassa virus-like particles led to a dramatic increase in vascular permeability in a viral load-dependent manner. Using this platform, we showed that Fibrin-derived peptide FX06 can be used to suppress the vascular integrity loss. This simple chip-based model proved promising in the assessment of disease severity and provides an easy-to-use platform for future investigation of Lassa pathogenesis and drug development in a human-like setting.

Human Organs-on-Chips for Virology.

While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro.

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects.

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

Ebola Hemorrhagic Shock Syndrome-on-a-Chip.

Ebola virus, for which we lack effective countermeasures, causes hemorrhagic fever in humans, with significant case fatality rates. Lack of experimental human models for Ebola hemorrhagic fever is a major obstacle that hinders the development of treatment strategies. Here, we model the Ebola hemorrhagic syndrome in a microvessel-on-a-chip system and demonstrate its applicability to drug studies. Luminal infusion of Ebola virus-like particles leads to albumin leakage from the engineered vessels. The process is mediated by the Rho/ROCK pathway and is associated with cytoskeleton remodeling. Infusion of Ebola glycoprotein (GP1,2) generates a similar phenotype, indicating the key role of GP1,2 in this process. Finally, we measured the potency of a recently developed experimental drug FX06 and a novel drug candidate, melatonin, in phenotypic rescue. Our study confirms the effects of FX06 and identifies melatonin as an effective, safe, inexpensive therapeutic option that is worth investigating in animal models and human trials.

Single-Cell Screening of Tamoxifen Abundance and Effect Using Mass Spectrometry and Raman-Spectroscopy.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Live single cell mass spectrometry reveals cancer-specific metabolic profiles of circulating tumor cells.

Recently, there has been increased attention on the analysis of circulating tumor cells (CTCs), also known as liquid biopsy, owing to its potential benefits in cancer diagnosis and treatment. Circulating tumor cells are released from primary tumor lesions into the blood stream and eventually metastasize to distant body organs. However, a major hurdle with CTC analysis is their natural scarcity. Existing methods lack sensitivity, specificity, or reproducibility required in CTC characterization and detection. Here, we report untargeted molecular profiling of single CTCs obtained from gastric cancer and colorectal cancer patients, using live single cell mass spectrometry integrated with microfluidics-based cell enrichment techniques. Using this approach, we showed the difference in the metabolomic profile between CTCs originating from different cancer groups. Moreover, potential biomarkers were putatively annotated to be specific to each cancer type.

Live Single-Cell Mass Spectrometry (LSC-MS) for Plant Metabolomics.

Live single-cell mass spectrometry (LSC-MS) allows for the detection of hundreds to thousands of metabolite peaks acquired from a single plant cell within a few minutes. Plant cells are first observed under a stereomicroscope, a cell of interest is chosen, and then sampled using a metal-coated glass microcapillary for subsequent analysis. A few microliters of ionization solvent is then added to the rear end of the capillary followed by the introduction of the capillary's content directly into the mass spectrometer. High voltage is applied between the capillary and the mass spectrometer inlet to induce nanospray ionization. Metabolite structural confirmation is performed using tandem mass spectrometry analysis (MS/MS) and fragments are matched with MS/MS databases to predict metabolic pathways. This method enables swift and direct molecular detection and identification of specific metabolites from a single plant cell along with their localization within the cell, which will allow for comprehensive understanding of plant metabolomics on a single cell level.

Single-Cell Metabolomics.

The dynamics of a cell is always changing. Cells move, divide, communicate, adapt, and are always reacting to their surroundings non-synchronously. Currently, single-cell metabolomics has become the leading field in understanding the phenotypical variations between them, but sample volumes, low analyte concentrations, and validating gentle sample techniques have proven great barriers toward achieving accurate and complete metabolomics profiling. Certainly, advanced technologies such as nanodevices and microfluidic arrays are making great progress, and analytical techniques, such as matrix-assisted laser desorption ionization (MALDI), are gaining popularity with high-throughput methodology. Nevertheless, live single-cell mass spectrometry (LCSMS) values the sample quality and precision, turning once theoretical speculation into present-day applications in a variety of fields, including those of medicine, pharmaceutical, and agricultural industries. While there is still room for much improvement, it is clear that the metabolomics field is progressing toward analysis and discoveries at the single-cell level.

Quantitative Live Single-cell Mass Spectrometry with Spatial Evaluation by Three-Dimensional Holographic and Tomographic Laser Microscopy.

The locations and volumes of the contents of a single HepG2 cell were visualized under three-dimensional (3D) holographic and tomographic (HT) laser microscopy, colored by refractive index, not staining. After trapping the specific area of a target cell in a nanospray tip, quantification was performed by live single-cell mass spectrometry. Comparison of the HepG2 cells' before and after 3D-HT images allowed the inference of the precise volume and original location of the trapped cell contents. The total amount of a trapped molecule was estimated. The images also revealed morphological changes in the cell structure caused by the manipulation.

Direct Lipido-Metabolomics of Single Floating Cells for Analysis of Circulating Tumor Cells by Live Single-cell Mass Spectrometry.

Direct trapping of a single floating cell, i.e. a white blood cell from a drop of blood, within a nanospray tip was followed by super-sonication after the addition of ionization solvent. Molecular detection of an increased number of peaks with a higher intensity and a wider m/z range, which extends from metabolites to lipids, was acquired than of that without sonication. This method was applied to a few separated circulating tumor cells (CTC) from a neuroblastoma patient's blood to obtain their lipido-metabolomic molecular profile at the single cell level. In addition to vital molecules such as amino acids, catechol amine metabolites, which are specific to neuroblastoma, and drugs included in the patient's course of therapy were detected. This established "direct single-cell lipido-metabolomic method" seems to be useful for direct and wide range molecular detection not only for many live single-cells, but also for rare cells, such as CTCs, for future molecular diagnosis.