Tetramethyl Rhodamine Research Articles

Vulpinic acid, a lichen metabolite, emerges as a potential drug candidate in the therapy of oxidative stress-related diseases, such as atherosclerosis.

Vulpinic acid, a lichen compound, has been shown to have many beneficial effects and its medicinal value increases day by day. As in atherosclerosis, endothelial damage is the basis of many diseases. The aim of this study is to investigate the effects of vulpinic acid against oxidative stress damage induced by hydrogen peroxide (H2O2) in endothelial cells. In order to find the IC50 of H2O2 and the protective dose of vulpinic acid, methyl thiazolyldiphenyl tetrazolium bromide (MTT) assays were performed. The amount of reactive oxygen species (ROS) induced by H2O2 and the protective effects of vulpinic acid against ROS were examined by fluorometric DCF-DA kit. The effects of H2O2 and vulpinic acid on actin filaments were determined by tetramethyl rhodamine (TRITC)-phalloidin fluorescence staining. Expression of Tie2 proteins was immunocytochemically analyzed in H2O2- and vulpinic acid-treated cells. After 24 h, the IC50 was found to be 215 μM in HUVECs treated with H2O2. The most effective dose of vulpinic acid against H2O2-associated damage was found to be 15 μM. Vulpinic acid pretreatment was shown to reduce H2O2-induced ROS production significantly ( p < 0.05). It was shown that 215 μM of H2O2 caused actin fragmentation, cell shrinkage, and decrease in actin florescence intensity while vulpinic acid protected the cells from these damages. It was found that Tie2 immunoreactivity was decreased in H2O2-treated groups and vulpinic acid pretreatment reduced the expression of this protein. In conclusion, vulpinic acid decreases H2O2-induced oxidative stress and oxidative stress-related damages in HUVECs. It may be drug candidate in the therapy of atherosclerosis.

Influence of Glyphaea brevis twig extract on nucleus, tight junctions and expression of inhibin-β, stem cell factor, and androgen binding protein in TM4 Sertoli cells

Objective: To examine the influence of Glyphaea (G.) brevis twig extract on the mitochondrial dehydrogenase activity, integrity of the tight junctions between adjacent cells, mitochondria, apoptosis, nucleus and expression of inhibin-s, stem cell factor, and androgen binding protein in TM4 Sertoli cells. Methods: TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells. TM4 Sertoli cells were exposed to G. brevis twig extract (0.1, 1.0, 10.0, 100.0, or 1 000.0μg/mL) for 24, 48 and 72 h. Parameters studied included cell viability [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay], mitochondrial membrane potential (tetra methyl rhodamine ethyl ester dye), transepithelial electrical resistance, apoptosis (Annexin V Alexa Fluor®488/propidium iodide assay) and mRNA expression (quantitative reverse transcription polymerase chain reaction). Results: G. brevis twig extract had no cytotoxic impact on cell viability, thus, considerably increasing the activity of mitochondrial dehydrogenase enzyme after 24 and 72 h exposure. Transepithelial electrical resistance values revealed substantial (P Conclusions: G. brevis twig extract may increase the secretory roles of TM4 Sertoli cells, cells proliferation, as well as cell-cell tight junction integrity. Thus, G. brevis twig may enhance spermatogenesis.

Using optical tweezers to construct an upconversion luminescent resonance energy transfer analytical platform

We report a new upconversion nanoparticles (UCNPs) based luminescent resonance energy transfer (LRET) analytical platform by making use of optical tweezers technology. The LRET model is designed by simultaneously conjugating Yb3+ and Er3+ co-doped UCNPs (as the donors) and tetramethyl rhodamine (TAMRA) molecules (as the acceptors) on microspheres to fabricate complex microspheres. Upon a single complex microsphere entering the three-dimensional potential well formed with a tightly focused 980 nm Guassian-shaped laser beam, it is optically trapped and concurrently the upconversion emission is excited, whereby the donor signals are transferred to the acceptors. As a proof-of-concept investigation, microRNA-21 sequences are selected as the targets, by which the distance between the two perfectly matched luminophors is controlled to several nanometers via nucleic acid hybridization. Without the involvement of luminescence amplification strategies, the proposed single microsphere based LRET method shows highly competitive sensitivity with a limit of detection down to 114 fM and satisfactory specificity towards microRNAs detection. Moreover, its practical working ability is demonstrated by credibly quantifying the absolute contents of miRNA-21 sequences in three cancer cell lines and even tracing the targets in as few as 100 cancer cells. Thus, this favorable analytical methodology provides an alternative for bioassays and holds certain potential in biomedical applications.

Transcription Factor Eb Is Required for Macropinocytosis-Mediated Growth Recovery of Nutrient-Deprived Kras-Mutant Cells.

Macropinocytosis is a regulated form of endocytosis that mediates the nonselective uptake of nutrients to support growth under nutrient-deprived conditions. KRAS-mutant cancer cells upregulate macropinocytosis to import extracellular proteins, which subsequently undergo proteolytic degradation in the lysosome. Although transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and function, its role in the degradation of extracellular protein from macropinocytosis in KRAS-mutant cells has not previously been elucidated. In this study, we investigated the role of TFEB in the recovery of macropinocytosis-mediated mTORC1 activity and cell growth under nutrient depletion. Mouse embryonic fibroblasts (MEFs) expressing KrasG12D and KRAS-mutant human cancer cells took up markedly higher levels of tetramethylrhodamine (TMR)-dextran than the corresponding wild-type cells. siRNA-mediated inhibition of TFEB did not influence extracellular TMR-dextran uptake, but significantly attenuated lysosomal degradation of extracellular protein. Bovine serum albumin (BSA) treatment restored p-S6K levels and cell proliferation suppressed by leucine deprivation, and these effects were blocked by siTFEB. Collectively, our results show that TFEB plays a role in macropinocytosis-mediated KRAS-mutant cell growth under nutrient deprivation by promoting lysosomal degradation of extracellular proteins.

10 - Electron transport chain inhibitors alter membrane potential and induce mitochondrial fragmentation in vascular smooth muscle cells: implications for bioenergetic measurements

Mitochondrial metabolism relies on membrane potential (ΔΨm) for effective electron transport across respiratory complexes and a filamentous mitochondrial network. Oxidative metabolism is typically measured via bioenergetic assays utilizing electron transport chain inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor). Bioenergetic effects of these compounds are well-established, but effects on ΔΨm and morphology in vascular smooth muscle cells (VSMC) remains largely unknown. Pulmonary hypertension (PH) is complex vasculopathy characterized by excess cell proliferation and a metabolic defect – PH VSMC preferentially use aerobic glycolysis for energy production, having experienced a metabolic switch similar to other proliferative diseases, including cancer. Using normal and PH patient pulmonary artery smooth muscle cells (hPASMC), mitochondrial metabolism was measured via micropolarimetry using oligomycin, FCCP, Antimycin A and rotenone. Mitochondrial morphology was assessed pre- and post-inhibitor treatment by confocal imaging of MitoTracker Deep Red-loaded hPASMC, and subsequent application of a machine learning algorithm to predict mitochondrial shape and calculate volume. ΔΨm was measured pre- and post- inhibitor treatment by flow cytometry of tetramethylrhodamine (TMRE)-loaded hPASMC. Compared to PH, normal hPASMC exhibit significantly higher baseline oxidative metabolism. PH PASMC display significant mitochondrial fragmentation (~60% intermediate/fragmented); normal PASMC are highly fused (~75% filamentous). ETC inhibitors induce fragmentation in normal PASMC, significantly reducing filamentous network in favor of intermediate rods. Fragmentation is further enhanced in ETC-inhibited PH PASMC (>80% fragmented/intermediate). ETC inhibitors also induce a 10-50% reduction of ΔΨm in normal PASMC. PH PASMC are comparatively hyperpolarized, and largely resistant to inhibitor-induced depolarization, suggesting the underlying metabolic defect in PH PASMC is independent of ΔΨm. ETC inhibitors used to study oxidative metabolism have off-target effects in normal VSMC, causing membrane depolarization and fragmentation. In PH patients, the mitochondrial network is destabilized and sensitive to further fragmentation by ETC inhibition, but this fragmentation is independent of membrane potential.

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method.

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Dye Aggregate-Mediated Self-Assembly of Bacteriophage Bioconjugates.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

In Vivo Two-Photon Voltage Imaging with Sulfonated Rhodamine Dyes.

Optical methods that rely on fluorescence for mapping changes in neuronal membrane potential in the brains of awake animals provide a powerful way to interrogate the activity of neurons that underlie neural computations ranging from sensation and perception to learning and memory. To achieve this goal, fluorescent indicators should be bright, highly sensitive to small changes in membrane potential, nontoxic, and excitable with infrared light. We report a new class of fluorescent, voltage-sensitive dyes: sulfonated rhodamine voltage reporters (sRhoVR), synthetic fluorophores with high voltage sensitivity, excellent two-photon performance, and compatibility in intact mouse brains. sRhoVR dyes are based on a tetramethyl rhodamine fluorophore coupled to a phenylenevinylene molecular wire/diethyl aniline voltage-sensitive domain. When applied to cells, sRhoVR dyes localize to the plasma membrane and respond to membrane depolarization with a fluorescence increase. The best of the new dyes, sRhoVR 1, displays a 44% ΔF/F increase in fluorescence per 100 mV change, emits at 570 nm, and possesses excellent two-photon absorption of approximately 200 GM at 840 nm. sRhoVR 1 can detect action potentials in cultured rat hippocampal neurons under both single- and two-photon illumination with sufficient speed and sensitivity to report on action potentials in single trials, without perturbing underlying physiology or membrane properties. The combination of speed, sensitivity, and brightness under two-photon illumination makes sRhoVR 1 a promising candidate for in vivo imaging in intact brains. We show sRhoVR powerfully complements electrode-based modes of neuronal activity recording in the mouse brain by recording neuronal transmembrane potentials from the neuropil of layer 2/3 of the mouse barrel cortex in concert with extracellularly recorded local field potentials (LFPs). sRhoVR imaging reveals robust depolarization in response to whisker stimulation; concurrent electrode recordings reveal negative deflections in the LFP recording, consistent with the canonical thalamocortical response. Importantly, sRhoVR 1 can be applied in mice with chronic optical windows, presaging its utility in dissecting and resolving voltage dynamics using two-photon functional imaging in awake, behaving animals.

In-vitro antitumor activity of new quaternary phosphonium salts, derivatives of 3-hydroxypyridine.

This work presents the results of in-vitro biological activity studies of three novel anticancer agents, phosphonium salts based on the 3-hydroxypyridine scaffold, including one derivative of 4-deoxypyridoxine. Proliferation and viability of cells treated with these compounds was assessed by the colony formation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Effects of the compounds on apoptosis and cell cycle were studied by flow cytometry using annexin V-FITC/propidium iodide and propidium iodide staining, respectively. The influence of the compounds on mitochondrial membrane potential and intracellular reactive oxygen species was evaluated using tetramethyl rhodamine ethyl and DCFHA staining. Western blot analysis was used to study the changes in the expression of Bcl-xL, Bax, and caspase-3 apoptotic proteins. The treatment of ovarian adenocarcinoma cells OVCAR-4 with the tested compounds inhibited the growth and induced cell cycle arrest in the G1 phase. 3-Hydroxypyridine derivatives induced apoptosis by hyperexpression of Bax and caspase-3, whereas 4-deoxypyridoxine derivative induced cell death partly by reactive oxygen species generation and caspase-3 hyperexpression. These results indicate that the quaternary phosphonium salts studied represent potential therapeutic agents for the treatment of ovarian cancer.

Direct fluorescence anisotropy approach for aflatoxin B1 detection and affinity binding study by using single tetramethylrhodamine labeled aptamer

The discovery of aptamers for aflatoxin B1 (AFB1), one of toxic carcinogens, has allowed to develop aptamer-based sensors and assays for aflatoxin. In this work, we reported a direct fluorescence anisotropy (FA) assay for investigation of aptamer-AFB1 binding and detection of AFB1 with the aptamer having single tetramethylrhodamine (TMR) label on a specific site. From a series of labeling sites of a 50-mer aptamer, we screened out the aptamer with TMR labeling at the 26th T, capable of generating good and large FA-decreasing response to AFB1. By using the T26-labeled 50-mer aptamer probe in FA analysis, we determined the affinity and selectivity of aptamer, and identified the crucial region of aptamer and optimum experimental conditions for strong binding. The aptamer could be further truncated to as short as 26 nucleotides in length, and this shorter aptamer possessed a simple stem-loop secondary structure and retained good binding affinity. Nucleotides in the loop region of the aptamer were conserved and important for affinity recognition. We achieved FA detection of AFB1 with a detection limit about 2 nM by using the TMR-labeled aptamer probe. The cross reactivity of aflatoxin B1, aflatoxin B2, aflatoxin M1, aflatoxin M2, aflatoxin G1, and aflatoxin G2 with aptamer were estimated to be 100%, 61%, 23%, 21%, 6.3%, 6.5%, respectively. The aptamer probe presented good selectivity over other mycotoxins and showed potential in complex sample analysis. This study of affinity binding between aptamer and aflatoxins will be helpful for developing other aptamer-based assays and sensors for aflatoxins.

TRANSCRIPTOME PROFILE OF NICOTINIC RECEPTOR–LINKED, BETA AMYLOID-INDUCED NEUROTOXICITY

Alzheimer's disease (AD) is a significant neurodegenerative disorder of the elderly. In order to develop potential therapeutic strategies for the management of this order, it is very critical to obtain a better understanding of the global gene changes underlying the progression of AD pathogenesis. In this study, we used RNA-seq technology to analyze global differential gene expression in the model neuroblastoma cell line NG108-15, transfected with α4β2 nAChRs, and treated or untreated with full-length beta amyloid (Aβ1-42), to gain insights into the underlying mechanisms of beta amyloid-induced neurotoxicity. Cells were treated with media or 100nM of Aβ1-42 for 3 days, followed by extraction and purification of their RNA. Multiplexed RNA-seq libraries were prepared from the cellular RNA and paired-end 100-bp sequencing was conducted using an Illumina HiSeq 2500 sequencer. Fold changes in the expression of transcripts (and hence, genes) between untreated and Aβ-treated samples were determined. Ingenuity pathway analysis (IPA) was employed to determine the cellular pathways. Treatment with Aβ1-42 in α4β2 nAChR-transfected cells differentially modulated the expression of 15 prominent genes as compared to that of untreated cells. Most notably, chrna4 and rcan3 (Regulator of Calcineurin) were upregulated 3-fold and 2-fold, respectively. The differential gene expression data identified by RNA-seq was validated by real time PCR, western blot and immunocytochemistry. Additionally, we used Cas9/CRISPR gene-editing system to knock out Rcan3. The sorted cells were further treated or not with Aβ1-42 for 3 days and the impact of loss of Rcan3 was examined in terms of oxidative stress, mitochondrial membrane potential and cell viability. The absence of Rcan3 was expected to reduce the extent of oxidative stress, as measured by counting reactive oxygen species (ROS)-positive cells and mitochondrial membrane potential, as determined by tetramethyl rhodamine ethyl ester (TMRE) staining, on exposure to beta amyloid. This is the first study to conduct an entire transcriptome profile of model nerve cells expressing a defined high-affinity target receptor, namely nAChR, for beta amyloid. Characterizing the genes associated with calcium signaling, specifically Rcan3 after beta amyloid treatment will have significant impact on developing neuroprotective agents to reduce or prevent neuronal death in AD patients.

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Photoactivatable Rhodamine Spiroamides and Diazoketones Decorated with "Universal Hydrophilizer" or Hydroxyl Groups.

Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.

P-186 - Cellular electrophile damage by 2-chlorohexadecanal in cardiomyocytes

Background Myeloperoxidase (MPO) activity increases in myocardial infarction due to neutrophil infiltration. MPO together with H2O2 and Cl- leads to the formation of HOCl, which induces plasmalogen modification. HOCl attacks the vinylether bond of plasmalogens inducing formation of alpha-chlorofatty aldehydes, like 2-chlorohexadecanal (2-ClHDA). 2-ClHDA impacts protein function by covalent modification, triggering cytotoxic responses. Methods To study 2-ClHDA-mediated protein modification in cardiomyocytes, click-chemistry with proteomic approaches was used. Therefore, an alkyne containing bioortholog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA), was synthesized. Protein lysates of 2-ClHDyA-modified cardiomyocytes were clicked with tetramethylrhodamine (TAMRA) azide and separated by 2-DE. TAMRA-positive spots were trypsinized and identified by LC-MS/MS. Results During these analyses 51 2-ClHDyA-modified proteins were identified. Those proteins were mainly attributed to cellular processes involved in stress response, the cytoskeleton, and enzymes responsible for energy metabolism. These results suggest that cellular damage following myocardial infarction could be caused by impaired protein function due to modification by 2-ClHDA.

Endogenous Membrane Receptor Labeling by Reactive Cytokines and Growth Factors to Chase Their Dynamics in Live Cells

Summary Cytokine and growth factor receptors localized on cell membranes play key roles in many biological events. Because the dynamic behaviors of receptors are deeply involved in cytokine- and growth-factor-dependent signaling pathways, reliable techniques for real-time imaging of receptors in live cells are strongly desired. Here, we describe a method of covalently labeling and imaging membrane receptors in live cells by using chemically reactive cytokines and growth factors coupled with 4-dimethylaminopyridine (DMAP), an organocatalyst that facilitates acyl transfer. Construction of the DMAP cytokines and growth factors relies on non-covalent coordination chemistry between an oligo-histidine tag and dimeric Ni(II)-nitrilotriacetate in an in situ manner. This supramolecular approach is relatively simple and flexible and can be applied to labeling not only transiently but also endogenously expressed receptors. Moreover, given the benefit of this traceless labeling of the receptors, our technique allows real-time imaging of the ligand-induced dynamics of endogenously expressed receptors in live cells.

Hollow carbon dots labeled with FITC or TRITC for use in fluorescent cellular imaging.

Hollow carbon dots (HCDs) were prepared by a solvothermal method and conjugated to either tetramethyl rhodamine isothiocyanate (TRITC) or fluorescein-5-isothiocyanate (FITC). This resulted in HCDs with bright red or green fluorescence, with excitation/emission peaks at 550/580 and 491/520nm, respectively. The nanocomposites are well water-soluble, remarkably photostable and biocompatible. In addition,the fluorescence of the composites is more stable in a reactive oxygen environment thanthe free dyes. Confocal images indicate that the nanoparticles quickly enter A549 cells and mainly accumulate in the cytoplasm. The wavelength of functionalized HCDs can be regulating via coupling the HCDs to different dyes. These results demonstrate that these composite materials can be very promising reagents for biological labeling and imaging. Graphical abstract Schematic of the preparation of hollow carbon dots conjugated to tetramethyl rhodamine isothiocyanate (RHCDs)by solvothermal method. The material is water-soluble, remarkably photostable and biocompatible. It was applied to cellular labeling and imaging.

Metaboloptics: Visualization of the tumor functional landscape via metabolic and vascular imaging

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO2 (“Warburg effect”), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO2, indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor’s adaptive behavior.

A simple method for determining the ligand affinity toward a zinc-enzyme model by using a TAMRA/TAMRA interaction.

Thiolate coordination to zinc(ii) ions occurs widely in such functional biomolecules as zinc enzymes or zinc finger proteins. Here, we introduce a simple method for determining the affinity of ligands toward the zinc-enzyme active-center model tetramethylrhodamine (TAMRA)-labeled 1,4,7,10-tetraazacyclododecane (cyclen)-zinc(ii) complex (TAMRA-ZnL). The 1 : 1 complexation of TAMRA-labeled cysteine (TAMRA-Cys) with TAMRA-ZnL (each at 2.5 μM), in which the TAMRA moieties approach one another closely, induces remarkable changes in the visible absorption and fluorescence spectra at pH 7.4 and 25 °C. The 1 : 1 complex formation constant (K = [thiolate-bound zinc(ii) complex]/[uncomplexed TAMRA-ZnL][uncomplexed TAMRA-Cys], M-1) was determined to be 106.7 M-1 from a Job's plot of the absorbances at 552 nm. By a ligand-competition method with the 1 : 1 complexation equilibrium, analogous K values for thiol-containing ligands, such as N-acetyl-l-cysteine, l-glutathione, and N-acetyl-l-cysteinamide, were evaluated to have similar values of about 104 M-1. As a result of the ligand affinities to TAMRA-ZnL, nonlabeled zinc(ii)-cyclen induced remarkable stabilization of the reduced form of l-glutathione and a cysteine-containing enolase peptide to aerial oxidation in aqueous solution at pH 7.4 and 25 °C.

Drug release kinetics of temperature sensitive liposomes measured at high-temporal resolution with a millifluidic device

Purpose: Current release assays have inadequate temporal resolution ( ∼ 10 s) to characterise temperature sensitive liposomes (TSL) designed for intravascular triggered drug release, where release within the first few seconds is relevant for drug delivery.Materials and methods: We developed a novel release assay based on a millifluidic device. A 500 µm capillary tube was heated by a temperature-controlled Peltier element. A TSL solution encapsulating a fluorescent compound was pumped through the tube, producing a fluorescence gradient along the tube due to TSL release. Release kinetics were measured by analysing fluorescence images of the tube. We measured three TSL formulations: traditional TSL (DPPC:DSPC:DSPE-PEF2000,80:15:5), MSPC-LTSL (DPPC:MSPC:DSPE-PEG2000,85:10:5) and MPPC-LTSL (DPPC:MMPC:PEF2000,86:10:4). TSL were loaded with either carboxyfluorescein (CF), Calcein, tetramethylrhodamine (TMR) or doxorubicin (Dox). TSL were diluted in one of the four buffers: phosphate buffered saline (PBS), 10% bovine serum albumin (BSA) solution, foetal bovine serum (FBS) or human plasma. Release was measured between 37–45 °C.Results: The millifluidic device allowed measurement of release kinetics within the first few seconds at ∼5 ms temporal resolution. Dox had the fastest release and highest release %, followed by CF, Calcein and TMR. Of the four buffers, release was fastest in human plasma, followed by FBS, BSA and PBS.Conclusions: The millifluidic device allows measurement of TSL release at unprecedented temporal resolution, thus allowing adequate characterisation of TSL release at time scales relevant for intravascular triggered drug release. The type of buffer and encapsulated compound significantly affect release kinetics and need to be considered when designing and evaluating novel TSL-drug combinations.

Investigating endogenous µ-opioid receptors in human keratinocytes as pharmacological targets using novel fluorescent ligand.

Opioids in skin function during stress response, regeneration, ageing and, particularly in regulating sensation. In chronic pruritus, topical treatment with Naltrexone changes μ-opioid receptor (μ-OR) localization to relieve itch. The molecular mechanisms behind the effects of Naltrexone on μ-OR function in reduction of itching behavior has not been studied. There is an immediate need to understand the endogenous complexity of μ-OR dynamics in normal and pathological skin conditions. Here we evaluate real-time behavior of μ-OR-Endomorphine complexes in the presence of agonist and antagonists. The μ-OR ligand Endomorphine-1 (EM) was conjugated to the fluorescent dye Tetramethylrhodamine (TAMRA) to investigate the effects of agonist and antagonists in N/TERT-1 keratinocytes. The cellular localization of the EM-TAMRA was followed through time resolved confocal microscopy and population analysis was performed by flow cytometry. The in vitro analyses demonstrate fast internalization and trafficking of the endogenous EM-TAMRA-μ-OR interactions in a qualitative manner. Competition with Endomorphine-1, Naltrexone and CTOP show both canonical and non-canonical effects in basal and differentiated keratinocytes. Acute and chronic treatment with Naltrexone and Endomorphine-1 increases EM-TAMRA binding to skin cells. Although Naltrexone is clinically effective in relieving itch, the mechanisms behind re-distribution of μ-ORs during clinical treatments are not known. Our study has given insight into cellular mechanisms of μ-OR ligand-receptor interactions after opioid agonist and antagonist treatments in vitro. These findings potentially offer opportunities in using novel treatment strategies for skin and peripheral sensory disorders.