Aberrant migration features in primary skin fibroblasts of Huntington's disease patients hold potential for unraveling disease progression using an image based machine learning tool.

Huntington's disease (HD) is a complex neurodegenerative disorder with considerable heterogeneity in clinical manifestations. While CAG repeat length is a known predictor of disease severity, this heterogeneity suggests the involvement of additional genetic and environmental factors. Previously we revealed that HD primary fibroblasts exhibit unique features, including distinct nuclear morphology and perturbed actin cap, resembling characteristics seen in Hutchinson-Gilford Progeria Syndrome (HGPS). This study establishes a link between actin cap deficiency and cell motility in HD, which correlates with the HD patient disease severity. Here, we examined single-cell motility imaging features in HD primary fibroblasts to explore in depth the relationship between cell migration patterns and their respective HD patients' clinical severity status (premanifest, mild and severe). The single-cell analysis revealed a decline in overall cell motility in correlation with HD severity, being most prominent in severe HD subgroup and HGPS. Moreover, we identified seven distinct spatial clusters of cell migration in all groups, which their proportion varies within each group becoming a significant HD severity classifier between HD subgroups. Next, we investigated the relationship between Lamin B1 expression, serving as nuclear envelope morphology marker, and cell motility finding that changes in Lamin B1 levels are associated with specific motility patterns within HD subgroups. Based on these data we present an accurate machine learning classifier offering comprehensive exploration of cellular migration patterns and disease severity markers for future accurate drug evaluation opening new opportunities for personalized treatment approaches in this challenging disorder.

Corrigendum: Applying new approach methodologies to assess next-generation tobacco and nicotine products.

[This corrects the article DOI: 10.3389/ftox.2024.1376118.].

Sensitive Detection of Genotoxic Substances in Complex Food Matrices by Multiparametric High-Content Analysis.

Genotoxic substances widely exist in the environment and the food supply, posing serious health risks due to their potential to induce DNA damage and cancer. Traditional genotoxicity assays, while valuable, are limited by insufficient sensitivity, specificity, and efficiency, particularly when applied to complex food matrices. This study introduces a multiparametric high-content analysis (HCA) for the detection of genotoxic substances in complex food matrices. The developed assay measures three genotoxic biomarkers, including γ-H2AX, p-H3, and RAD51, which enhances the sensitivity and accuracy of genotoxicity screening. Moreover, the assay effectively distinguishes genotoxic compounds with different modes of action, which not only offers a more comprehensive assessment of DNA damage and the cellular response to genotoxic stress but also provides new insights into the exploration of genotoxicity mechanisms. Notably, the five tested food matrices, including coffee, tea, pak choi, spinach, and tomato, were found not to interfere with the detection of these biomarkers under proper dilution ratios, validating the robustness and reliability of the assay for the screening of genotoxic compounds in the food industry. The integration of multiple biomarkers with HCA provides an efficient method for detecting and assessing genotoxic substances in the food supply, with potential applications in toxicology research and food safety.

Applying new approach methodologies to assess next-generation tobacco and nicotine products.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and "roadmaps" to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

Stable knockdown of Drp1 improves retinoic acid-BDNF-induced neuronal differentiation through global transcriptomic changes and results in reduced phosphorylation of ERK1/2 independently of DUSP1 and 6.

Background: Dynamin-related protein Drp1 -a major mitochondrial fission protein- is widely distributed in the central nervous system and plays a crucial role in regulating mitochondrial dynamics, specifically mitochondrial fission and the organelle's shaping. Upregulated Drp1 function may contribute to the pathological progression of neurodegenerative diseases by dysregulating mitochondrial fission/ fusion. The study aims to investigate the effects of Drp1 on retinoic acid-BDNF-induced (RA-BDNF) neuronal differentiation and mitochondrial network reorganization in SH-SY5Y neuroblastoma cells. Methods: We generated an SH-SY5Y cell line with stably depleted Drp1 (shDrp1). We applied RNA sequencing and analysis to study changes in gene expression upon stable Drp1 knockdown. We visualized the mitochondria by transmission electron microscopy and used high-content confocal imaging to characterize and analyze cell morphology changes and mitochondrial network reorganization during neuronal differentiation. Results: shDrp1 cells exhibited fused mitochondrial ultrastructure with perinuclear clustering. Stable knockdown of Drp1 resulted in the upregulation of genes involved in nervous system development. High content analysis showed improved neurite outgrowth, segmentation, and extremities in differentiated shDrp1 cells. Neuronal differentiation was associated with a significant reduction in ERK1/2 phosphorylation, and ERK1/2 phosphorylation was independent of the dual specificity phosphatases DUSP1/6 in shDrp1 cells. Differentiated control underwent mitochondrial morphology remodeling, whereas differentiated shDrp1 cells retained the highly fused mitochondria and developed long, elongated structures. The shDrp1 cells responded to specific apoptotic stimuli like control in vitro, suggesting that Drp1 is not a prerequisite for apoptosis in SH-SY5Y cells. Moreover, Drp1 downregulation reduced the formation of toxic mHtt aggregates in vitro. Discussion: Our results indicate that Drp1 silencing enhances RA-BDNF-induced neuronal differentiation by promoting transcriptional and mitochondrial network changes in undifferentiated cells. We also demonstrate that the suppression of Drp1 reduces toxic mHtt aggregate formation in vitro, suggesting protection against neurotoxicity. Thus, Drp1 may be an attractive target for further investigation in future strategies to combat neurodegenerative diseases.

High-content analysis identified synergistic drug interactions between INK128, an mTOR inhibitor, and HDAC inhibitors in a non-small cell lung cancer cell line.

BACKGROUND: The development of drug resistance is a major cause of cancer therapy failures. To inhibit drug resistance, multiple drugs are often treated together as a combinatorial therapy. In particular, synergistic drug combinations, which kill cancer cells at a lower concentration, guarantee a better prognosis and fewer side effects in cancer patients. Many studies have sought out synergistic combinations by small-scale function-based targeted growth assays or large-scale nontargeted growth assays, but their discoveries are always challenging due to technical problems such as a large number of possible test combinations. METHODS: To address this issue, we carried out a medium-scale optical drug synergy screening in a non-small cell lung cancer cell line and further investigated individual drug interactions in combination drug responses by high-content image analysis. Optical high-content analysis of cellular responses has recently attracted much interest in the field of drug discovery, functional genomics, and toxicology. Here, we adopted a similar approach to study combinatorial drug responses. RESULTS: By examining all possible combinations of 12 drug compounds in 6 different drug classes, such as mTOR inhibitors, HDAC inhibitors, HSP90 inhibitors, MT inhibitors, DNA inhibitors, and proteasome inhibitors, we successfully identified synergism between INK128, an mTOR inhibitor, and HDAC inhibitors, which has also been reported elsewhere. Our high-content analysis further showed that HDAC inhibitors, HSP90 inhibitors, and proteasome inhibitors played a dominant role in combinatorial drug responses when they were mixed with MT inhibitors, DNA inhibitors, or mTOR inhibitors, suggesting that recessive drugs could be less prioritized as components of multidrug cocktails. CONCLUSIONS: In conclusion, our optical drug screening platform efficiently identified synergistic drug combinations in a non-small cell lung cancer cell line, and our high-content analysis further revealed how individual drugs in the drug mix interact with each other to generate combinatorial drug response.

MAARS Software for Automatic and Quantitative Analysis of Mitotic Progression.

In this chapter, we describe a software called MAARS (Mitotic Analysis And Recording System) that enables automatic and quantitative analysis of mitotic progression on an open-source platform. This computer-assisted analysis of cell division allows the unbiased acquisition of multiple parameters such as cell shape or size, metaphase or anaphase delays, as well as various mitotic abnormalities. This chapter describes the power of such an expert system to highlight the complexity of the mechanisms required to prevent mitotic chromosome segregation errors, leading to aneuploidy.

A novel ERß high throughput microscopy platform for testing endocrine disrupting chemicals.

In this study we present an inducible biosensor model for the Estrogen Receptor Beta (ERß), GFP-ERß:PRL-HeLa, a single-cell-based high throughput (HT) in vitro assay that allows direct visualization and measurement of GFP-tagged ERß binding to ER-specific DNA response elements (EREs), ERß-induced chromatin remodeling, and monitor transcriptional alterations via mRNA fluorescence in situ hybridization for a prolactin (PRL)-dsRED2 reporter gene. The model was used to accurately (Z' = 0.58-0.8) differentiate ERß-selective ligands from ERα ligands when treated with a panel of selective agonists and antagonists. Next, we tested an Environmental Protection Agency (EPA)-provided set of 45 estrogenic reference chemicals with known ERα in vivo activity and identified several that activated ERß as well, with varying sensitivity, including a subset that is completely novel. We then used an orthogonal ERE-containing transgenic zebrafish (ZF) model to cross validate ERß and ERα selective activities at the organism level. Using this environmentally relevant ZF assay, some compounds were confirmed to have ERß activity, validating the GFP-ERß:PRL-HeLa assay as a screening tool for potential ERß active endocrine disruptors (EDCs). These data demonstrate the value of sensitive multiplex mechanistic data gathered by the GFP-ERß:PRL-HeLa assay coupled with an orthogonal zebrafish model to rapidly identify environmentally relevant ERß EDCs and improve upon currently available screening tools for this understudied nuclear receptor.

Quantitative assessment of mitochondrial morphology relevant for studies on cellular health and environmental toxicity.

Mitochondria are essential organelles that play crucial roles in cellular energy metabolism, calcium signaling and apoptosis. Their importance in tissue homeostasis and stress responses, combined to their ability to transition between various structural and functional states, make them excellent organelles for monitoring cellular health. Quantitative assessment of mitochondrial morphology can therefore provide valuable insights into environmentally-induced cell damage. High-content screening (HCS) provides a powerful tool for analyzing organelles and cellular substructures. We developed a fully automated and miniaturized HCS wet-plus-dry pipeline (MITOMATICS) exploiting mitochondrial morphology as a marker for monitoring cellular health or damage. MITOMATICS uses an in-house, proprietary software (MitoRadar) to enable fast, exhaustive and cost-effective analysis of mitochondrial morphology and its inherent diversity in live cells. We applied our pipeline and big data analytics software to assess the mitotoxicity of selected chemicals, using the mitochondrial uncoupler CCCP as an internal control. Six different pesticides (inhibiting complexes I, II and III of the mitochondrial respiratory chain) were tested as individual compounds and five other pesticides present locally in Occitanie (Southern France) were assessed in combination to determine acute mitotoxicity. Our results show that the assayed pesticides exhibit specific signatures when used as single compounds or chemical mixtures and that they function synergistically to impact mitochondrial architecture. Study of environment-induced mitochondrial damage has the potential to open new fields in mechanistic toxicology, currently underexplored by regulatory toxicology and exposome research. Such exploration could inform health policy guidelines and foster pharmacological intervention, water, air and soil pollution control and food safety.

Use of Fluorescent Chemical Probes in the Study of Toll-like Receptors (TLRs) Trafficking.

Fluorescent chemical probes are used nowadays as a chemical resource to study the physiology and pharmacology of several important endogenous receptors. Different fluorescent groups have been coupled with known ligands of these receptors, allowing the visualization of their localization and trafficking. One of the most important molecular players of innate immunity and inflammation are the Toll-Like Receptors (TLRs). These Pattern-Recognition Receptors (PRR) have as natural ligands microbial-derived pathogen-associated molecular patterns (PAMPs) and also endogenous molecules called danger-associated molecular patterns (DAMPs). These ligands activate TLRs to start a response that will determine the host's protection and overall cell survival but can also lead to chronic inflammation and autoimmune syndromes. TLRs action is tightly related to their subcellular localization and trafficking. Understanding this trafficking phenomenon can enlighten critical molecular pathways that might allow to decipher the causes of different diseases. In this chapter, the study of function, localization and trafficking of TLRs through the use of chemical probes will be discussed. Furthermore, an example protocol of the use of fluorescent chemical probes to study TLR4 trafficking using high-content analysis will be described.

Quantitation of macropinocytosis in glioblastoma based on high-content analysis.

BACKGROUND: Macropinocytosis is a pathway utilized for the internalization of extracellular fluid, albumin and dissolved molecules. Assessing macropinocytosis has been challenging in the past because the combination of manual acquisition and visual evaluation of images is laborious, making this type of assessment difficult for high-throughput applications. Therefore, there is a need to develop sensitive and specific macropinocytosis evaluation methods. METHODS: This paper proposed a quantitative and time-saving method for macropinocytosis detection based on high-content analysis (HCA). Additionally, cell proliferation was evaluated using CCK8 test. RESULTS: The term "macropinosome index" was defined to estimate macropinocytosis and allow comparisons between different cell lines and treatments. Furthermore, we demonstrated that macropinocytosis can promote glioblastoma (GBM) cell survival under L-glutamine (L-Gln)-deficient conditions that resemble the tumour microenvironment. CONCLUSIONS: HCA represents a novel, nonsubjective and high-throughput assay for macropinocytosis assessment. In addition, L-Gln deprivation increased the macropinosome index in GBM cells, suggesting that this process may be used to design GBM therapies. AVAILABILITY OF DATA AND MATERIALS: The datasets supporting the conclusions of this article are included within the article and its supplementary materials.

Lysophosphatidylcholine-DHA Specifically Induces Cytotoxic Effects of the MDA-MB-231 Human Breast Cancer Cell Line In Vitro-Comparative Effects with Other Lipids Containing DHA.

Docosahexaenoic acid (DHA, C22:6 ω-3) is a dietary polyunsaturated fatty acid that has an important role in human health. Epidemiological studies linked a high intake of DHA to a reduced risk of certain cancers. Recently, attention focused on how the lipid carrier in which DHA is delivered, i.e., esterified on acylglycerols, phospholipids, or free, affects its biological effects. However, studies comparing the effects of these different forms for DHA supply to cancer cells in vitro are limited. In this study, the effect of free DHA and five lipids carrying one to three DHA chains (LPC-DHA, PC-DHA, MAG-DHA, DAG-DHA and TAG-DHA) on the viability of the MDA-MB-231 breast cancer cell line was compared. Our results revealed a strong structure-function relationship of DHA-carrying lipids on the viability of MDA-MB-231 cells. Glycerophosphocholine-based lipids are the most effective DHA carriers in reducing the viability of MDA-MB-231 cells, with LPC-DHA being more effective (IC50 = 23.7 µM) than PC-DHA (IC50 = 67 µM). The other tested lipids are less toxic (MAG-DHA, free DHA) or even not toxic (DAG-DHA, TAG-DHA) under our conditions. Investigating the mechanism of cell death induced by LPC-DHA revealed increased oxidative stress and membrane cell damage.

Modeling drug-induced liver injury and screening for anti-hepatofibrotic compounds using human PSC-derived organoids.

Preclinical models that can accurately predict the toxicity and efficacy of candidate drugs to human liver tissue are in urgent need. Human liver organoid (HLO) derived from human pluripotent stem cells offers a possible solution. Herein, we generated HLOs, and demonstrated the utility of these HLOs in modeling a diversity of phenotypes associated with drug-induced liver injury (DILI), including steatosis, fibrosis, and immune responses. Phenotypic changes in HLOs after treatment with tool compounds such as acetaminophen, fialuridine, methotrexate, or TAK-875 showed high concordance with human clinical data in drug safety testings. Moreover, HLOs were able to model liver fibrogenesis induced by TGFß or LPS treatment. We further devised a high-content analysis system, and established a high-throughput anti-fibrosis drug screening system using HLOs. SD208 and Imatinib were identified that can significantly suppress fibrogenesis induced by TGFß, LPS, or methotrexate. Taken together, our studies demonstrated the potential applications of HLOs in drug safety testing and anti-fibrotic drug screening.

High-content analysis of testicular toxicity of BPA and its selected analogs in mouse spermatogonial, Sertoli cells, and Leydig cells revealed BPAF induced unique multinucleation phenotype associated with the increased DNA synthesis.

Bisphenol A is an endocrine disruptor that has been shown to have testicular toxicity in animal models. Its structural analog, including bisphenol S (BPS), bisphenol AF (BPAF), and tetrabromobisphenol A (TBBPA) have been introduced to the market as BPA alternatives. Previously, we developed high-content analysis (HCA) assays and applied machine learning to compare the testicular toxicity of BPA and its analogs in spermatogonial cells and testicular cell co-culture models. There are diverse cell populations in the testis to support spermatogenesis, but their cell type-specific toxicities are still not clear. The purpose of this study is to examine the selective toxicity of BPA, BPS), BPAF, and TBBPA on these testicular cells, including Sertoli cells, Leydig cells, and spermatogonia cells. We developed a high-content image-based single-cell analysis and measured a broad spectrum of adverse endpoints related to the development of reproductive toxicology, including cell number, nuclear morphology, DNA synthesis, cell cycle progression, early DNA damage response, cytoskeleton structure, DNA methylation status, and autophagy. We introduced an HCA index and spectrum to reveal multiple HCA parameters and observed distinct toxicity profiling of BPA and its analogs among three testicular types. The HCA spectrum shows the dynamic, chemical-specific, dose-dependent changes of each HCA parameter. Each chemical displayed a unique dose-dependent profile within each type of cell. All three types of cells showed the highest response to BPAF at 10 µM across all endpoints measured. BPAF targeted spermatogonial cell (C18) more significantly at 5 µM. BPS more likely targeted Sertoli cell (TM4) and Leydig cell (TM3) and less at spermatogonia cells. TBBPA targeted spermatogonia, Sertoli cells, and less at TM3 cells. BPA is mainly targeted at TM4, followed by TM3 cells, and less at spermatogonial cells. Most importantly, we observed that BPAF induced a dose-dependent increase in spermatogonia cells, not in Sertoli and Leydig cells. In summary, our current HCA assays revealed the cell-type-specific toxicities of BPA and its analogs in different testicular cells. Multinucleation induced by BPAF, along with increased DNA damage and synthesis at low doses, could possibly have a profound long-term effect on reproductive systems.

Microglial Phagocytosis/Cell Health High-Content Assay.

We report a microglial phagocytosis/cell health high-content assay that has been used to test small molecule chemical probes and support our drug discovery projects targeting microglia for Alzheimer's disease therapy. The assay measures phagocytosis and cell health (cell count and nuclear intensity) simultaneously in 384-well plates processed with an automatic liquid handler. The mix-and-read live cell imaging assay is highly reproducible with capacity to meet drug discovery research needs. Assay procedures take 4 days including plating cells, treating cells, adding pHrodo-myelin/membrane debris to cells for phagocytosis, staining cell nuclei before performing high-content imaging, and analysis. Three selected parameters are measured from cells: 1) mean total fluorescence intensity per cell of pHrodo-myelin/membrane debris in phagocytosis vesicles to quantify phagocytosis; 2) cell counts per well (measuring compound effects on proliferation and cell death); and 3) average nuclear intensity (measuring compound induced apoptosis). The assay has been used on HMC3 cells (an immortalized human microglial cell line), BV2 cells (an immortalized mouse microglial cell line), and primary microglia isolated from mouse brains. Simultaneous measurements of phagocytosis and cell health allow for the distinction of compound effects on regulation of phagocytosis from cellular stress/toxicity related changes, a distinguishing feature of the assay. The combination of cell counts and nuclear intensity as indicators of cell health is also an effective way to measure cell stress and compound cytotoxicity, which may have broad applications as simultaneous profiling measurements for other phenotypic assays. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Microglial phagocytosis/cell health high-content assay protocol Support Protocol: Procedures to isolate myelin/membrane debris from mouse brain and label with pHrodo.

Perturbed actin cap as a new personalized biomarker in primary fibroblasts of Huntington's disease patients.

Primary fibroblasts from patient's skin biopsies are directly isolated without any alteration in the genome, retaining in culture conditions their endogenous cellular characteristics and biochemical properties. The aim of this study was to identify a distinctive cell phenotype for potential drug evaluation in fibroblasts from Huntington's Disease (HD) patients, using image-based high content analysis. We show that HD fibroblasts have a distinctive nuclear morphology associated with a nuclear actin cap deficiency. This in turn affects cell motility in a similar manner to fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients used as known actin cap deficient cells. Moreover, treatment of the HD cells with either Latrunculin B, used to disrupt actin cap formation, or the antioxidant agent Mitoquinone, used to improve mitochondrial activity, show expected opposite effects on actin cap associated morphological features and cell motility. Deep data analysis allows strong cluster classification within HD cells according to patients' disease severity score which is distinct from HGPS and matching controls supporting that actin cap is a biomarker in HD patients' cells correlated with HD severity status that could be modulated by pharmacological agents as tool for personalized drug evaluation.

High-Resolution Bacterial Cytological Profiling Reveals Intrapopulation Morphological Variations upon Antibiotic Exposure.

Phenotypic heterogeneity is crucial to bacterial survival and could provide insights into the mechanism of action (MOA) of antibiotics, especially those with polypharmacological actions. Although phenotypic changes among individual cells could be detected by existing profiling methods, due to the data complexity, only population average data were commonly used, thereby overlooking the heterogeneity. In this study, we developed a high-resolution bacterial cytological profiling method that can capture morphological variations of bacteria upon antibiotic treatment. With an unprecedented single-cell resolution, this method classifies morphological changes of individual cells into known MOAs with an overall accuracy above 90%. We next showed that combinations of two antibiotics induce altered cell morphologies that are either unique or similar to that of an antibiotic in the combinations. With these combinatorial profiles, this method successfully revealed multiple cytological changes caused by a natural product-derived compound that, by itself, is inactive against Acinetobacter baumannii but synergistically exerts its multiple antibacterial activities in the presence of colistin. The findings have paved the way for future single-cell profiling in bacteria and have highlighted previously underappreciated intrapopulation variations caused by antibiotic perturbation.

Repurposing the tyrosine kinase inhibitor nilotinib for use against intracellular multidrug-resistant Salmonella Typhimurium.

BACKGROUND/PURPOSE: The increasing incidence of infections caused by multidrug-resistant Salmonella enterica has become a serious threat to global public health. Here, we found that the tyrosine kinase inhibitor nilotinib exhibits antibacterial activity against intracellular S. enterica serovar Typhimurium in RAW264.7 macrophages. Thus, we aimed to pharmacologically exploit the anti-intracellular Salmonella activity of nilotinib and to elucidate its mechanism of action. METHODS: The antibacterial activity of the compounds was assessed by high-content analysis (HCA) and intracellular CFU, minimum inhibitory concentration (MIC), and bacterial growth assays. The cytotoxicity of the compounds was evaluated by HCA and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assays. The levels of cellular AMPK, phospho-AMPK, Atg7 and ß-actin were determined by immunoblotting. RESULTS: The screen identified two small molecule compounds (SCT1101 and SCT1104) with potent activity against intracellular S. Typhimurium. Moreover, SCT1101 and SCT1104 enhanced the efficacy of ciprofloxacin and cefixime against intracellular S. Typhimurium. However, only SCT1101 exhibited activity against intracellular MDR and fluoroquinolone-resistant S. Typhimurium isolates. Subsequent mechanistic studies showed that neither of these nilotinib derivatives increased the phospho-AMPK level in RAW264.7 cells. Neither the AMPK inhibitor compound C nor SBI-0206965 reversed the inhibitory effects of SCT1101 and SCT1104 on intracellular Salmonella. Furthermore, neither blockade of autophagy by 3-MA nor shRNA-mediated knockdown of Atg7 protein expression in RAW264.7 cells affected the antibacterial activity of SCT1101 and SCT1104. CONCLUSION: The structure of nilotinib could be used to develop novel therapeutics for controlling MDR S. Typhimurium infections.

A High-Throughput Method to Profile Protein Liquid-Liquid Phase Separation.

Protein liquid-liquid phase separation (LLPS) plays an essential role in the dynamic assembly of various membraneless compartments, which fulfill different biological functions in cells. Numerous proteins were found to undergo LLPS in different conditions. However, a general approach to systemically identify and compare the LLPS ability of different proteins is lacking. Here, we introduce a high-throughput protein phase separation (HiPPS) profiling method to evaluate the LLPS ability of proteins using a combination of crystallization robot/manual mixing mode and high-content analysis system. This method enables us to rapidly and comprehensively explore the LLPS behavior of each individual protein as well as mixture of different proteins.

High content analysis of in vitro alveolar macrophage responses can provide mechanistic insight for inhaled product safety assessment.

Assessing the safety of inhaled substances in the alveolar region of the lung requires an understanding of how the respired material interacts with both physical and immunological barriers. Human alveolar-like macrophages in vitro provide a platform to assess the immunological response in the airways and may better inform the understanding of a response to an inhaled challenge being adaptive or adverse. The aim of this study was to determine if a morphometric phenotyping approach could discriminate between different inhaled nicotine products and indicate the potential mechanism of toxicity of a substance. Cigarette smoke (CS) and e-liquids extracted into cell culture medium were applied to human alveolar-like macrophages in mono-culture (ImmuONE™) and co-culture (ImmuLUNG™) to test the hypothesis. Phenotype profiling of cell responses was highly reproducible and clearly distinguished the different responses to CS and e-liquids. Whilst the phenotypes of untreated macrophages were similar regardless of culture condition, macrophages cultured in the presence of epithelial cells were more sensitive to CS-induced changes related to cell size and vacuolation processes. This technique demonstrated phenotypical observations typical for CS exposure and indicative of the established mechanisms of toxicity. The technique provides a rapid screening approach to determine detailed immunological responses in the airways which can be linked to potentially adverse pathways and support inhalation safety assessment.