scholarly journals A carbon nanotube optical reporter maps endolysosomal lipid flux

2017 ◽  
Author(s):  
Prakrit V. Jena ◽  
Daniel Roxbury ◽  
Thomas V. Galassi ◽  
Leila Akkari ◽  
Christopher P. Horoszko ◽  
...  
Keyword(s):  
Carbon Nanotube ◽  
Single Cell ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Live Cells ◽  
Lysosomal Storage ◽  
Drug Induced ◽  
Spatially Resolved ◽  
Order Of Magnitude ◽  
Multiple Drugs

ABSTRACTLipid accumulation within the lumen of endolysosomal vesicles is observed in various pathologies including atherosclerosis, liver disease, neurological disorders, lysosomal storage disorders, and cancer. Current methods cannot measure lipid flux specifically within the lysosomal lumen of live cells. We developed an optical reporter, composed of a photoluminescent carbon nanotube of a single chirality, which responds to lipid accumulation via modulation of the nanotube’s optical bandgap. The engineered nanomaterial, composed of short-single stranded DNA and a single nanotube chirality, localizes exclusively to the lumen of endolysosomal organelles without adversely affecting cell viability or proliferation, or organelle morphology, integrity, or function. The emission wavelength of the reporter can be spatially resolved from within the endolysosomal lumen to generate quantitative maps of lipid content in live cells. Endolysosomal lipid accumulation in cell lines, an example of drug-induced phospholipidosis (DIPL), was observed for multiple drugs in macrophages, and measurements of patient-derived Niemann-Pick type C fibroblasts identified lipid accumulation and phenotypic reversal of this lysosomal storage disease. Single-cell measurements using the reporter discerned sub-cellular differences in equilibrium lipid content, illuminating significant intracellular heterogeneity among endolysosomal organelles of differentiating bone marrow-derived monocytes. Single-cell kinetics of lipoprotein-derived cholesterol accumulation within macrophages revealed rates that differed among cells by an order of magnitude. This carbon nanotube optical reporter of endolysosomal lipid content in live cells confers new capabilities for drug development processes and the investigation of lipid-linked diseases.

scholarly journals An optical nanoreporter of endolysosomal lipid accumulation reveals enduring effects of diet on hepatic macrophages in vivo

2018 ◽  
Vol 10 (461) ◽  
pp. eaar2680 ◽  
Author(s):  
Thomas V. Galassi ◽  
Prakrit V. Jena ◽  
Janki Shah ◽  
Geyou Ao ◽  
Elizabeth Molitor ◽  
...  
Keyword(s):  
Drug Development ◽  
Lipid Accumulation ◽  
Near Infrared ◽  
Infrared Emission ◽  
Normal Diet ◽  
Lysosomal Storage ◽  
Preclinical Research ◽  
Drug Induced ◽  
Hepatic Macrophages

The abnormal accumulation of lipids within the endolysosomal lumen occurs in many conditions, including lysosomal storage disorders, atherosclerosis, nonalcoholic fatty liver disease (NAFLD), and drug-induced phospholipidosis. Current methods cannot monitor endolysosomal lipid content in vivo, hindering preclinical drug development and research into the mechanisms linking endolysosomal lipid accumulation to disease progression. We developed a single-walled carbon nanotube–based optical reporter that noninvasively measures endolysosomal lipid accumulation via bandgap modulation of its intrinsic near-infrared emission. The reporter detected lipid accumulation in Niemann-Pick disease, atherosclerosis, and NAFLD models in vivo. By applying the reporter to the study of NAFLD, we found that elevated lipid quantities in hepatic macrophages caused by a high-fat diet persist long after reverting to a normal diet. The reporter dynamically monitored endolysosomal lipid accumulation in vivo over time scales ranging from minutes to weeks, indicating its potential to accelerate preclinical research and drug development processes.

scholarly journals Hyperspectral Counting of Multiplexed Nanoparticle Emitters in Single Cells and Organelles

2021 ◽  
Author(s):  
Prakrit V Jena ◽  
Mitchell A Gravely ◽  
Christian Cupo ◽  
Mohammad Moein Safaee ◽  
Daniel Roxbury ◽  
...  
Keyword(s):  
Carbon Nanotube ◽  
Single Cell ◽  
Single Cells ◽  
Cellular Structures ◽  
Live Cells ◽  
Tissue Cell ◽  
Significant Heterogeneity ◽  
The Subject ◽  
Non Gaussian ◽  
Accurate Quantification

Nanomaterials are the subject of a range of biomedical, commercial, and environmental investigations involving measurements in living cells and tissues. Accurate quantification of nanomaterials, at the tissue, cell, and organelle levels, is often difficult, however, in part due to their inhomogeneity. Here, we propose a method that uses the diverse optical properties of a nanomaterial preparation in order to improve quantification at the single-cell and organelle level. We developed 'hyperspectral counting', which employs diffraction-limited imaging via hyperspectral microscopy of a diverse set of nanomaterial emitters, to estimate nanomaterial counts in live cells and sub-cellular structures. A mathematical model was developed, and Monte Carlo simulations were employed, to improve the accuracy of these estimates, enabling quantification with single-cell and single-endosome resolution. We applied this nanometrology technique to identify an upper-limit of the rate of uptake into cells -approximately 3,000 particles endocytosed within 30 minutes. In contrast, conventional ROI counting results in a 230% undercount. The method identified significant heterogeneity and a broad non-Gaussian distribution of carbon nanotube uptake within cells. For example, while a particular cell contained an average of 1 nanotube per endosome, the heterogenous distribution resulted in over 7 nanotubes localizing within some endosomes, substantially changing the accounting of subcellular nanoparticle concentration distributions. This work presents a method to quantify cellular and subcellular concentrations of a heterogeneous carbon nanotube reference material, with implications for nanotoxicology, drug/gene delivery, and nanosensor fields.

scholarly journals Single-cell transcriptomic analysis of mIHC images via antigen mapping

2021 ◽  
Vol 7 (10) ◽  
pp. eabc5464
Author(s):  
Kiya W. Govek ◽  
Emma C. Troisi ◽  
Zhen Miao ◽  
Rachael G. Aubin ◽  
Steven Woodhouse ◽  
...  
Keyword(s):  
Single Cell ◽  
Spatial Patterns ◽  
Cell Types ◽  
Level Of Detail ◽  
Cell Populations ◽  
Sequencing Data ◽  
Spatially Resolved ◽  
Murine Spleen ◽  
Single Cell Rna Sequencing ◽  
Antibody Panel

Highly multiplexed immunohistochemistry (mIHC) enables the staining and quantification of dozens of antigens in a tissue section with single-cell resolution. However, annotating cell populations that differ little in the profiled antigens or for which the antibody panel does not include specific markers is challenging. To overcome this obstacle, we have developed an approach for enriching mIHC images with single-cell RNA sequencing data, building upon recent experimental procedures for augmenting single-cell transcriptomes with concurrent antigen measurements. Spatially-resolved Transcriptomics via Epitope Anchoring (STvEA) performs transcriptome-guided annotation of highly multiplexed cytometry datasets. It increases the level of detail in histological analyses by enabling the systematic annotation of nuanced cell populations, spatial patterns of transcription, and interactions between cell types. We demonstrate the utility of STvEA by uncovering the architecture of poorly characterized cell types in the murine spleen using published cytometry and mIHC data of this organ.

scholarly journals TAMI-45. PHENOGENOMIC CHARACTERIZATION OF IMMUNOMODULATORY PURINERGIC SIGNALING IN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii222-ii223
Author(s):  
Shannon Coy ◽  
Rumana Rashid ◽  
Sylwia Stopka ◽  
Jia-Ren Lin ◽  
Philipp Euskirchen ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Single Cell ◽  
Purinergic Signaling ◽  
Progression Free Survival ◽  
Tissue Imaging ◽  
Phenotypic Properties ◽  
Spatially Resolved ◽  
Therapeutic Benefits ◽  
Adenosine Concentration ◽  
Cd73 Expression

Abstract INTRODUCTION Purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within tumors such as glioblastoma, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate immunosuppressive adenosine-rich environments. However, the composition, architecture, and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype are poorly characterized. METHODS We quantified CD73 expression by immunohistochemistry in a cohort of CNS tumors [meningiomas(n=222), gliomas(n=244), ependymomas(n=44), medulloblastomas(n=24), and craniopharyngiomas(n=38)]. We used publicly-available single-cell RNA-seq data and 36-marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastoma resections to characterize CD39 and CD73-expressing populations, define the immune architecture and tumor cell-states at single cell resolution, and identify markers of clinical outcome. We used mass spectrometry imaging (MALDI-MSI) to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (n=10). RESULTS CD73 exhibited strong expression in a subset of gliomas and meningiomas but was typically not expressed in ependymomas or medulloblastomas. CD73 expression correlated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq and t-CyCIF in glioblastoma showed CD73 expression in tumor cells, and CD39 expression in macrophages and endothelial cells. MALDI-MSI showed significantly greater adenosine concentrations (3.5-fold;p=0.04) in glioblastomas with high CD73 expression. scRNA-seq showed direct correlations between stem-like mRNA expression, proliferation, and CD73 expression in DIPG. CD73 expression significantly correlated with EGFR amplification, interferon signaling, and PD-L1 expression in glioblastoma. CONCLUSIONS Phenogenomic analysis of purinergic immunomodulatory signaling revealed significant interplay between CD73 activity and genotype, adenosine concentration, differentiation-state, clinical outcome, and possible interaction between CD39-positive macrophages and CD73-positive neoplastic cells. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling.

High-content screening of drug-induced cardiotoxicity using quantitative single cell imaging cytometry on microfluidic device

Lab on a Chip ◽  
2011 ◽  
Vol 11 (1) ◽  
pp. 104-114 ◽  
Author(s):  
Min Jung Kim ◽  
Su Chul Lee ◽  
Sukdeb Pal ◽  
Eunyoung Han ◽  
Joon Myong Song
Keyword(s):  
Single Cell ◽  
Microfluidic Device ◽  
Cell Imaging ◽  
High Content Screening ◽  
Drug Induced ◽  
Single Cell Imaging ◽  
Imaging Cytometry

scholarly journals Drug-Induced Hepatic Injury in Newborns and Children in Intensive Care Unit: A Retrospective Study of China

2021 ◽  
Author(s):  
Ling Ye ◽  
Chengxian Guo ◽  
Zeying Feng ◽  
Longjian Huang ◽  
Chengjun Guo ◽  
...  
Keyword(s):  
Intensive Care ◽  
Liver Injury ◽  
Hepatic Injury ◽  
Sodium Succinate ◽  
Drug Combinations ◽  
Fat Emulsion ◽  
Drug Induced ◽  
Multiple Drugs ◽  
Enzyme Metabolism ◽  
Long Chain

Abstract Purpose Drug-induced liver injury (DILI) is a common adverse reaction in the clinic; however, there are relatively few reports of DILI in critically ill newborns and children. Making use of the Pediatric Intensive Care database (PIC), this study identifies which drugs are related to DILI in neonates and children in China. Methods Using the PIC, we screened for patients whose liver was suspected of being injured by drugs during hospitalization. The medicine they used was then assessed by the Roussel Uclaf Causality Assessment Method (RUCAM). We also collated drug combinations that may affect CYP enzyme metabolism, which may be one of the mechanisms that lead to DILI. Results A total of 13,449 patients were assessed, of whom 77 newborns and 261 children were finally included. The main type of liver injury in neonates was mixed (83.1%), while children’s hepatic injury types were mostly distributed between hepatocellular (59.4%) and cholestatic (28.4%). In terms of the assessment by the RUCAM, in newborns, the drugs that were most considered to cause or associated with hepatic injury comprised medium and long chain fat emulsion (17%), sodium glycerophosphate (12%) and meropenem (9%); while omeprazole (11%), methylprednisolone sodium succinate (10%) and meropenem (8%) are the primary culprit of DILI in children. Drug combinations that may affect CYP enzyme metabolism frequently seen in neonates are omeprazole + budesonide (16.9%), dexamethasone + midazolam (10.4%) and midazolam + sildenafil (10.4%). In children, the commonly used drug combinations are fentanyl + midazolam (20.7%), ibuprofen + furosemide (18.4%) and diazepam + omeprazole (15.3%). Conclusions The drugs that have been found to have hepatotoxicity (meropenem, medium and long chain fat emulsion, ibuprofen.etc.) are also related to DILI in newborns and children. When giving these drugs to newborns and children, physicians need to be more cautious. Also, pay attention to the effect on CYP 450 enzymes when using multiple drugs at the same time.

scholarly journals Drug-induced QT interval prolongation in cancer patients

2011 ◽  
Vol 4 (4) ◽  
pp. 223
Author(s):  
Torben K. Becker ◽  
Sai-Ching J. Yeung
Keyword(s):  
Cancer Patients ◽  
Qt Interval ◽  
Alkylating Agents ◽  
Torsade De Pointes ◽  
Interval Prolongation ◽  
Drug Induced ◽  
Vascular Disruption ◽  
Qt Interval Prolongation ◽  
Increased Risk ◽  
Multiple Drugs

Cancer patients are at an increased risk for QT interval prolongation and subsequent potentially fatal Torsade de pointes tachycardia due to the multiple drugs used for treatment of malignancies and the associated symptoms and complications. Based on a systematic review of the literature, this article analyzes the risk for prolongation of the QT interval with antineoplastic agents and commonly used concomitant drugs. This includes anthracyclines, fluorouracil, alkylating agents, and new molecularly targeted therapeutics, such as vascular disruption agents. Medications used in the supportive care can also prolong QT intervals, such as methadone, 5-HT3-antagonists and antihistamines, some antibiotics, antifungals, and antivirals. We describe the presumed mechanism of QT interval prolongation, drug-specific considerations, as well as important clinical interactions. Multiple risk factors and drug–drug interactions increase this risk for dangerous arrhythmias. We propose a systematic approach to evaluate cancer patients for the risk of QT interval prolongation and how to prevent adverse effects.

scholarly journals Spatial and single-cell transcriptional landscape of human cerebellar development

2020 ◽  
Author(s):  
Kimberly A. Aldinger ◽  
Zach Thomson ◽  
Parthiv Haldipur ◽  
Mei Deng ◽  
Andrew E. Timms ◽  
...  
Keyword(s):  
Single Cell ◽  
Emotional Regulation ◽  
Cell Types ◽  
Molecular Organization ◽  
Cerebellar Development ◽  
Disease Genes ◽  
Spatially Resolved ◽  
Human Cerebellum ◽  
Spatial Composition ◽  
And Function

ABSTRACTCerebellar development and function require precise regulation of molecular and cellular programs to coordinate motor functions and integrate network signals required for cognition and emotional regulation. However, molecular understanding of human cerebellar development is limited. Here, we combined spatially resolved and single-cell transcriptomics to systematically map the molecular, cellular, and spatial composition of early and mid-gestational human cerebellum. This enabled us to transcriptionally profile major cell types and examine the dynamics of gene expression within cell types and lineages across development. The resulting ‘Developmental Cell Atlas of the Human Cerebellum’ demonstrates that the molecular organization of the cerebellar anlage reflects cytoarchitecturally distinct regions and developmentally transient cell types that are insufficiently captured in bulk transcriptional profiles. By mapping disease genes onto cell types, we implicate the dysregulation of specific cerebellar cell types, especially Purkinje cells, in pediatric and adult neurological disorders. These data provide a critical resource for understanding human cerebellar development with implications for the cellular basis of cerebellar diseases.

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