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Liquid chromatography-mass spectrometry (LC-MS) based metabolomics suffers from extended duty cycles and matrix-dependent quantitation. Chemical tags with 96 unique masses are reported, which alleviate the metabolomic workflow bottleneck and allow for absolute quantitation. A metabolic screen for carboxylic acids was performed on mammalian cells deprived of various nutrients and showed 24% RSD and analysis of 288 samples in 2 h.

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Discovery and identification of a new endogenous metabolite are typically hindered by requirements of large sample volumes and multistage purifications to guide synthesis of the standard. Presented here is a metabolomics platform that uses chemical tagging and tandem mass spectrometry to determine structure, direct synthesis, and confirm identity. Three new homocysteine metabolites are reported: N-succinyl homocysteine, 2-methyl-1,3-thiazinane-4-carboxylic acid (MTCA), and homolanthinone.

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Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation. Fragmentation of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) releases two trimethylamines and the acyl chains through neutral loss and generates a unique double cyclization constant mass reporter. Selective incorporation of isotopes onto the tag produces a six-plex set of isobaric reagents. For equivalent six-plex-labeled samples, <14% RSD was achieved, followed by a dynamic range of 1:10 without signal compression. Quantification of PCs/LPCs in human hepatic cancer cells was conducted as six-plex using data-dependent analysis tandem MS. We report a six-plex qualitative and quantitative isobaric tagging strategy expanding the limits of analyzing PCs/LPCs.

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DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

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BACKGROUND: Nonneuronal cells, including epithelial cells, can produce acetylcholine (ACh). Muscarinic ACh receptor antagonists are used clinically to treat asthma and other medical conditions; however, knowledge regarding the roles of ACh in type 2 immunity is limited. OBJECTIVE: Our aim was to investigate the roles of epithelial ACh in allergic immune responses. METHODS: Human bronchial epithelial (HBE) cells were cultured with allergen extracts, and their ACh production and IL-33 secretion were studied in vitro. To investigate immune responses in vivo, naive BALB/c mice were treated intranasally with different muscarinic ACh receptor antagonists and then exposed intranasally to allergens. RESULTS: At steady state, HBE cells expressed cellular components necessary for ACh production, including choline acetyltransferase and organic cation transporters. Exposure to allergens caused HBE cells to rapidly release ACh into the extracellular medium. Pharmacologic or small-interfering RNA-based blocking of ACh production or autocrine action through the M3 muscarinic ACh receptors in HBE cells suppressed allergen-induced ATP release, calcium mobilization, and extracellular secretion of IL-33. When naive mice were exposed to allergens, ACh was quickly released into the airway lumen. A series of clinical M3 muscarinic ACh receptor antagonists inhibited allergen-induced IL-33 secretion and innate type 2 immune response in the mouse airways. In a preclinical murine model of asthma, an ACh receptor antagonist suppressed allergen-induced airway inflammation and airway hyperreactivity. CONCLUSIONS: ACh is released quickly by airway epithelial cells on allergen exposure, and it plays an important role in type 2 immunity. The epithelial ACh system can be considered a therapeutic target in allergic airway diseases.

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OBJECTIVES: Circadian misalignment and sleep deprivation often occur in tandem, and both negatively impact glucose homeostasis and metabolic health. The present study employed a forced desynchrony protocol to examine the influence of extended wakefulness and circadian misalignment on hourly glucose levels. METHODS: Nine healthy adults (4F/5M; 26 ± 4years) completed a 31-day in-laboratory protocol. After three 24 hour baseline days with 8 hours scheduled sleep opportunities, participants were scheduled to 14 consecutive 42.85 hour sleep-wake cycles, with 28.57 hours extended wakefulness and 14.28 hours sleep opportunities each cycle. Blood was sampled hourly across the forced desynchrony and over 600 plasma samples per participant were analyzed for glucose levels. RESULTS: Both hours into the 42.85 hours forced desynchrony day and circadian phase modulated glucose levels (p < .0001). Glucose peaked after each meal during scheduled wakefulness and decreased during scheduled sleep/fasting. Glucose levels were, on average, lowest during the biological daytime and rose throughout the biological night, peaking in the biological morning. When analyzed separately for scheduled sleep vs. wakefulness, the peak timing of the circadian rhythm in glucose was later during sleep (p < .05). Glucose area under the curve levels increased rapidly from the beginning of the forced desynchrony protocol and were highest on the second forced desynchrony day (p < .01), returning towards forced desynchrony day 1 levels thereafter. CONCLUSIONS: These findings have important implications for understanding factors contributing to altered glucose metabolism during sleep loss and circadian misalignment, and for potential physiological adaptation of metabolism in healthy adults, who are increasingly exposed to such conditions in our society.

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DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described.

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Environmental allergens that interact with the airway epithelium can activate cellular stress pathways that lead to the release of danger signals known as alarmins. The mechanisms of alarmin release are distinct from damage-associated molecular patterns (DAMPs), which typically escape from cells after loss of plasma membrane integrity. Oxidative stress represents a form of allergen-induced cellular stress that stimulates oxidant-sensing mechanisms coupled to pathways, which facilitate alarmin mobilization and efflux across the plasma membrane. In this review, we highlight examples of alarmin release and discuss their roles in the initiation of type 2 immunity and allergic airway inflammation. In addition, we discuss the concept of alarmin amplification, where "primary" alarmins, which are directly released in response to a specific cellular stress, stimulate additional signaling pathways that lead to secretion of "secondary" alarmins that include proinflammatory cytokines, such as IL-33, as well as genomic and mitochondrial DNA that coordinate or amplify type 2 immunity. Accordingly, allergen-evoked cellular stress can elicit a hierarchy of alarmin signaling responses from the airway epithelium that trigger local innate immune reactions, impact adaptive immunity, and exacerbate diseases including asthma and other chronic inflammatory conditions that affect airway function.

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Isobaric tags typically leverage an a1 type fragmentation to produce constant mass reporter ions. While this motif allows for efficient reporter formation, isobaric tags lack structural diversity, which limits the number and type of isotopes that are synthetically available. Presented here are two examples of dual fragmentation isobaric tagging. The first example mimics the typical isobaric tag structure through trimethylamine neutral loss and cyclization. Subsequent fragmentation releases a constant mass reporter with high efficiency. This provides a route to create a variety of isobaric tags with regard to both the reporter and the balancer mass. The second example is a set of six-plex isobaric, thiol-reactive tags that produce constant mass reporters by a similar sequential fragmentation mechanism. A trimethylamine neutral loss allows for the incorporation of up to 13 total isotopes in the balancer region, while minimizing deuterium retention time shifts. A subsequent C-S bond cleavage produces a constant mass reporter in the low-mass region. The thiols investigated produced an average RSD of 14% and R2 of 0.98 when analyzed as a six-plex injection. Thiol metabolism was disrupted using the glutamyl-cysteine synthetase inhibitor buthionine sulfoximine (BSO). Endothelial cells were incubated with BSO and showed significant decreases in glutathione and cysteinyl-glycine compared to control. Overall, a new method to generate constant mass reporters using a dual fragmentation scheme is presented.

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BACKGROUND: Alternaria alternata and house dust mite exposure evokes IL-33 secretion from the airway epithelium, which functions as an alarmin to stimulate type 2 immunity. Extracellular DNA (eDNA) is also an alarmin that intensifies inflammation in cystic fibrosis, chronic obstructive pulmonary disease, and asthma. OBJECTIVE: We investigated the mechanisms underlying allergen-evoked DNA mobilization and release from the airway epithelium and determined the role of eDNA in type 2 immunity. METHODS: Human bronchial epithelial (hBE) cells were used to characterize allergen-induced DNA mobilization and extracellular release using comet assays to measure DNA fragmentation, Qubit double-stranded DNA assays to measure DNA release, and DNA sequencing to determine eDNA composition. Mice were used to investigate the role of eDNA in type 2 immunity. RESULTS: Alternaria extract rapidly induces mitochondrial and nuclear DNA release from human bronchial epithelial cells, whereas house dust mite extract induces mitochondrial DNA release. Caspase-3 is responsible for nuclear DNA fragmentation and becomes activated after cleavage by furin. Analysis of secreted nuclear DNA showed disproportionally higher amounts of promotor and exon sequences and lower intron and intergenic regions compared to predictions of random DNA fragmentation. In mice, Alternaria-induced type 2 immune responses were blocked by pretreatment with a DNA scavenger. In caspase-3-deficient mice, Alternaria-induced DNA release was suppressed. Furthermore, intranasal administration of mouse genomic DNA with Alternaria amplified secretion of IL-5 and IL-13 into bronchoalveolar lavage fluid while DNA alone had no effect. CONCLUSION: These findings highlight a novel, allergen-induced mechanism of rapid DNA release that amplifies type 2 immunity in airways.

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Isobaric labelling of fatty acids is complicated by chromatographic co-elution of double bond isomers. This produces contaminated spectra which can mask important biological changes. Here two derivatization strategies are combined to improve throughput and produce MS2 reporters which change mass depending on double bond position. A 6-plex isobaric tag is attached to the acid group, followed by the tosylation of the double bond using chloramine-T. These two derivatizations allowed for the chromatographic resolution of nearly all investigated isomers using a 3.5 minute ultrafast method. Further isomer differentiation is achieved upon fragmentation as reporter masses scale with the double bond location. This occurs by a dual-fragmentation route which reveals the isobaric labelling and fragments along the double bond of each analyte. These unique fragments allowed for accurate quantitation of co-isolated double bond isomers where traditional isobaric tags would experience ratio distortion. Saturated and monounsaturated fatty acids were characterized by this rapid 6-plex method and produced an average signal RSD of 9.3% and R2 of 0.99. The method was then used to characterize fatty acid dysregulation upon inhibition of stearoyl CoA desaturase with CAY10566.

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Isobaric labeling in mass spectrometry enables multiplexed absolute quantitation and high throughput, while minimizing full scan spectral complexity. Here, we use 4-plex isobaric labeling with a fixed positive charge tag to improve quantitation and throughput for polar carboxylic acid metabolites. The isobaric tag uses an isotope-encoded neutral loss to create mass-dependent reporters spaced 2 Da apart and was validated for both single- and double-tagged analytes. Tags were synthesized in-house using deuterated formaldehyde and methyl iodide in a total of four steps, producing cost-effective multiplexing. No chromatographic deuterium shifts were observed for single- or double-tagged analytes, producing consistent reporter ratios across each peak. Perfluoropentanoic acid was added to the sample to drastically increase retention of double-tagged analytes on a C18 column. Excess tag was scavenged and extracted using hexadecyl chloroformate after reaction completion. This allowed for removal of excess tag that typically causes ion suppression and column overloading. A total of 54 organic acids were investigated, producing an average linearity of 0.993, retention time relative standard deviation (RSD) of 0.58%, and intensity RSD of 12.1%. This method was used for absolute quantitation of acid metabolites comparing control and type 1 diabetic urine. Absolute quantitation of organic acids was achieved by using one isobaric lane for standards, thereby allowing for analysis of six urine samples in two injections. Quantified acids showed good agreement with previous work, and six significant changes were found. Overall, this method demonstrated 4-plex absolute quantitation of acids in a complex biological sample.

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Biological aldehydes are difficult to analyze by electrospray ionization mass spectrometry due to their poor proton affinity and low biological concentrations. Chemical derivatization with stable isotope tags is used here for sample multiplexing, increased throughput, improved signal intensity, and quantitation. Nine quaternary amine tags with mass differences as low as 0.0058 Da had no observable chromatographic shifts, small amounts of ion suppression, and minimal matrix effects. Low concentration perfluoropentanoic acid was used as an ion pairing reagent to improve the retention of derivatized aldehydes. Perfluoropentanoic acid addition showed an average of three-fold improvement in limits of detection, 50% reduction in peak width, and 2.5 fold increase in analyte retention. Analysis of fifteen tagged aldehydes yielded an average of 13 nM limit of detection, 9 %RSD, R2 of 0.995, and linear dynamic range of 40-1000 nM. In a single 20 min separation, absolute quantitative data was obtained for 11 reactive aldehydes across 8 aortic endothelial cell samples. High glucose treatment produced significant changes to malondialdehyde, decanal, and (2E)-hexadecenal. These changes are consistent with glucose-induced oxidative stress. This method demonstrates that neutron encoded tagging of aldehydes is suitable for the analysis of complex samples.

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Photodeoxygenation of dibenzothiophene S-oxide and its derivatives have been used to generate atomic oxygen [O(3P)] to examine its effect on proteins, nucleic acids, and lipids. The unique reactivity and selectivity of O(3P) have shown distinct oxidation products and outcomes in biomolecules and cell-based studies. To understand the scope of its global impact on the cell, we treated MDA-MB-231 cells with 2,8-diacetoxymethyldibenzothiophene S-oxide and UV-A light to produce O(3P) without targeting a specific cell organelle. Cellular responses to O(3P)-release were analyzed using cell viability and cell cycle phase determination assays. Cell death was observed when cells were treated with higher concentrations of sulfoxides and UV-A light. However, significant differences in cell cycle phases due to UV-A irradiation of the sulfoxide were not observed. We further performed RNA-Seq analysis to study the underlying biological processes at play, and while UV-irradiation itself influenced gene expression, there were 9 upregulated and 8 downregulated genes that could be attributed to photodeoxygenation.

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Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 µM), the VDAC-1 inhibitor, DIDS (100 µM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 µM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.

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Zika virus (ZIKV) is an arbovirus belonging to the flaviviridae family with a risk assessment that has been increasing in recent years and was labeled a global health emergency by the World Health Organization in 2016. There are currently no Food and Drug Administration-approved treatment options available for ZIKV, so expeditious development of treatment options is urgent. To expedite this process, an on-market drug, tamoxifen (TAM), was selected as a promising candidate for repurposing due to its wide range of biological activities and because it has already been shown to possess activity against hepatitis C virus, a flavivirus in a separate genus. Anti-ZIKV activity of TAM was assessed by compound screens using an infectious virus and mechanistic details were gleaned from time of addition and virucidal studies. TAM and an active metabolite, 4-hydroxytamoxifen (TAM-OH), both showed promising antiviral activity (EC50 ≈9 and 5 µM, respectively) in initial compound screening and up to 8-h postinfection, though the virucidal assay indicated that they do not possess any direct virucidal activity. Additionally, TAM was assessed for its activity against ZIKV in the human male germ cell line, SEM-1, due to the sexually transmitted nature of ZIKV owing to its extended survival times in germ cells. Virus titers show diminished replication of ZIKV over 7 days compared to controls. These data indicate that TAM has the potential to be repurposed as an anti-ZIKV therapeutic and warrants further investigation.

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Photodeoxygenation of Dibenzothiophene-S-oxide (DBTO) in UV-A light produces atomic oxygen [O(3P)] and the corresponding sulfide, dibenzothiophene (DBT). Recently, DBTO has been derivatized to study the effect of UV-A light-driven photodeoxygenation in lipids, proteins, and nucleic acids. In this study, two DBTO derivatives with triphenylphosphonium groups were synthesized to promote mitochondrial accumulation. The sulfone analogs of these derivatives were also synthesized and used as fluorescent mitochondrial dyes to assess localization in mitochondria of HeLa cells. These derivatives were then used to study the effect of photodeoxygenation on MDA-MB-231 breast cancer cell line using cell viability assays, cell cycle phase determination tests, and RNA-Seq analysis. The DBTO derivatives were found to significantly decrease cell viability only after UV-A irradiation as a result of generating corresponding sulfides that were found to significantly affect gene expression and cell cycle.

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Adenosine and uric acid (UA) play a pivotal role in lung diseases such as asthma and chronic obstructive pulmonary disease (COPD). In the present experiments, we measured adenosine synthesis from nicotinamide adenine dinucleotide (NAD+) in membranes prepared from wild type (WT) and CD38 knockout (CD38KO) mouse lungs, from cultured airway smooth muscle and epithelial cells, and in bronchoalveolar lavage fluid after airway challenge with epidemiologically relevant allergens. Adenosine was determined using an enzymatically coupled assay that produces ATP and is detected by luminescence. Uric acid was determined by ELISA. Exposure of cultured airway epithelial cells to Alternaria alternata extract caused significant nucleotide (NAD+ and ATP) release in the culture media. The addition of NAD+ to membranes prepared from WT mice resulted in faster generation of adenosine compared to membranes from CD38KO mice. Formation of adenosine from NAD+ affected UA and ATP concentrations, its main downstream molecules. Furthermore, NAD+ and adenosine concentrations in the bronchoalveolar lavage fluid decreased significantly following airway challenge with house-dust mite extract in WT but not in CD38KO mice. Thus, NAD+ is a significant source of adenosine and UA in the airways in mouse models of allergic airway disease, and the capacity for their generation from NAD+ is augmented by CD38, a major NADase with high affinity for NAD+. This novel non-canonical NAD+-adenosine-UA pathway that is triggered by allergens has not been previously described in the airways.

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KEY POINTS: Alternaria aeroallergens induce the release of ATP from human bronchial epithelial (HBE) cells by activating a conductive pathway involving voltage-dependent anion channel-1 (VDAC-1) and by exocytosis of ATP localized within membrane vesicles. Inhibition of VDAC-1 blocked Alternaria-evoked Ca2+ uptake across the plasma membrane of HBE cells and interleukin (IL)-33 release into the extracellular media. Reducing cholesterol content with a cholesterol scavenger (ß-methylcyclodextrin) or statin compound (simvastatin) blocked ATP and IL-33 release by lowering the expression of VDAC-1 in the plasma membrane. Pretreatment with simvastatin for 24 h also inhibited the increase in tight junction macromolecule permeability that occurs following Alternaria exposure. These results establish a novel role for VDAC-1 as a mechanism underlying ATP release induced by fungal allergens and suggests a possible therapeutic use for cholesterol lowering compounds in reducing Alternaria-stimulated allergic inflammation. ABSTRACT: Human bronchial epithelial (HBE) cells exposed to allergens derived from the common saprophytic fungus, Alternaria alternata release ATP, which in turn stimulates P2X7 receptor-mediated Ca2+ uptake across the plasma membrane. The subsequent increase in intracellular calcium concentration induces proteolytic processing and secretion of interleukin (IL)-33, a critical cytokine involved in the initiation of allergic airway inflammation. A major objective of the present study was to identify the mechanism responsible for conductive ATP release. The results show that pretreatment of HBE cells with inhibitors of the voltage-dependent anion channel-1 (VDAC-1) or treatment with a VDAC-1 selective blocking antibody or silencing mRNA expression of the channel by RNA interference, inhibit Alternaria-evoked ATP release. Moreover, inhibition of VDAC-1 channel activity or reducing protein expression blocked the secretion of IL-33. Similarly, reducing the cholesterol content of HBE cells with simvastatin or the cholesterol scavenger ß-methylcyclodextrin also blocked ATP release and IL-33 secretion by decreasing the level of VDAC-1 expression in the plasma membrane. In addition, simvastatin inhibited the increase in tight junction macromolecule permeability that was previously observed after Alternaria exposure. These results demonstrate a novel function for VDAC-1 as the conductive mechanism responsible for Alternaria-induced ATP release, an essential early step in the processing, mobilization and secretion of IL-33 by the airway epithelium. Furthermore, the simvastatin-evoked reduction of VDAC-1 expression in the plasma membrane, suggests the possibility that cholesterol lowering compounds may be beneficial in alleviating allergic airway inflammation induced by fungal allergens.

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Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission. Due to agreeable wavelengths of excitation and emission, a standard 4',6-diamindino-2-phenylindole (DAPI) filter set worked well with these dyes. After co-staining, it was confirmed that both DBTOO dyes localized in the plasma membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The novel dyes also displayed relatively low toxicity to the HeLa cells with IC50 between 10 and 100 µm. Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.

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