Random forest and live single-cell metabolomics reveal metabolic profiles of human macrophages upon polarization.

Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single-cell resolution. To address this knowledge gap, an untargeted live single-cell mass spectrometry-based metabolomic profiling coupled with a machine-learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single-cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single-cell metabolomics provided an in-depth profile of the metabolome of primary human M1 and M2 macrophages at the single-cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells.

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.

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.

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.

Ultra high performance liquid chromatography with tandem mass spectrometry for rapid separation and identification of main constituents in Chinese herbal formula Xuan Hu Suo San.

Xuan Hu Suo San (XHSS) is a traditional Chinese medicine that has been extensively applied in the treatment of osteoarthritis for many years, however, its chemical composition has not yet been elucidated. Thus, a rapid, efficient, and precise method based on ultra high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry was applied in both positive- and negative-ion modes to rapidly separate and identify the main chemical ingredients in XHSS for the first time. Finally, with the optimised separation and detection method, a total of 57 compounds were simultaneously separated within 13 min, among which 14 compounds were confirmed by comparing retention time and MS data with reference standards and others were tentatively identified by comparing with reference literatures. This rapid and sensitive approach is highly useful for the identification and characterisation of chemical constituents, and provides fundamental and extensive information supporting further metabolic and pharmacokinetic studies of XHSS.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Alternaria alternata.

Fungi of the Alternaria genus are associated with allergic diseases, with Alternaria alternata being one of the most prevalent species. A. alternata has been frequently reported as the etiologic agent of hypersensitivity pneumonitis, allergic rhinosinusitis, bronchial asthma, and other diseases. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay and a real-time PCR assay to detect low levels of A. alternata in herbal tea samples. The LAMP assay can detect as little as 3 pg/µL of A. alternata genomic DNA with high specificity. In addition, both the LAMP assay and the real-time PCR assay can be used for quantification of A. alternata. Although the newly developed LAMP assay is more rapid and specific in A. alternata identification, the real-time PCR assay is more precise in quantitation analysis.

Pharmacodynamic study on insomnia-curing effects of Shuangxia Decoction in Drosophila melanogaster.

The present study aimed to establish a pharmacodynamic method using the pySolo software to explore the influence of freeze-dried powders of Shuangxia Decoction (SXD) on the sleep of normal Drosophila melanogaster and the Drosophila melanogaster whose sleep was divested by light. The dose-effect and the time-effect relationships of SXD on sleep were examined. The effect-onset concentration of SXD was 0.25%, the plateau appeared at the concentration of 2.5% and the total sleep time showed a downtrend when the concentration was greater than 2.5%. The sleep time was the longest on the fourth day after SXD was given. The fruit fly sleep deprivation model was repeated by light stimulation at night. The middle dosage group (2.5%) had the best insomnia-curing effect. In conclusion, using the pySolo software, an approach for the pharmacodynamics study was established with Drosophila melanogaster as a model organism to determine the insomnia-curing effects of the traditional Chinese medicine (TCM). Our results demonstrated the reliability of this method. The freeze-dried powders of SXD could effectively improve the sleep quality of Drosophila melanogaster.

A Network Pharmacology Approach to Uncover the Pharmacological Mechanism of XuanHuSuo Powder on Osteoarthritis.

As the most familiar type of arthritis and a chronic illness of the joints, Osteoarthritis (OA) affects a great number of people on the global scale. XuanHuSuo powder (XHSP), a conventional herbal formula from China, has been extensively applied in OA treatment. Nonetheless, its pharmacological mechanism has not been completely expounded. In this research, a network pharmacology approach has been chosen to study the pharmacological mechanism of XHSP on OA, and the pharmacology networks were established based on the relationship between four herbs found in XHSP, compound targets, and OA targets. The pathway enrichment analysis revealed that the significant bioprocess networks of XHSP on OA were regulation of inflammation, interleukin-1ß (IL-1ß) production and nitric oxide (NO) biosynthetic process, response to cytokine or estrogen stimuli, and antiapoptosis. These effects have not been reported previously. The comprehensive network pharmacology approach developed by our research has revealed, for the first time, a connection between four herbs found in XHSP, corresponding compound targets, and OA pathway systems that are conducive to expanding the clinical application of XHSP. The proposed network pharmacology approach could be a promising complementary method by which researchers might better evaluate multitarget or multicomponent drugs on a systematic level.