Intracellular flow cytometric lipid analysis - a multiparametric system to assess distinct lipid classes in live cells.

The lipid content of mammalian cells varies greatly between cell type. Current methods for analysing lipid components of cells are technically challenging and destructive. Here, we report a facile, inexpensive method to identify lipid content - intracellular flow cytometric lipid analysis (IFCLA). Distinct lipid classes can be distinguished by Nile Blue fluorescence, Nile Red fluorescence or violet autofluorescence. Nile Blue is fluorescent in the presence of unsaturated fatty acids with a carbon chain length greater than 16. Cis-configured fatty acids induce greater Nile Blue fluorescence than their trans-configured counterparts. In contrast, Nile Red exhibits greatest fluorescence in the presence of cholesterol, cholesteryl esters, some triglycerides and phospholipids. Multiparametric spanning-tree progression analysis for density-normalized events (SPADE) analysis of hepatic cellular lipid distribution, including vitamin A autofluorescence, is presented. This flow cytometric system allows for the rapid, inexpensive and non-destructive identification of lipid content, and highlights the differences in lipid biology between cell types by imaging and flow cytometry.

Microwave-Assisted Catalytic Method for a Green Synthesis of Amides Directly from Amines and Carboxylic Acids.

Amide bonds are among the most interesting and abundant molecules of life and products of the chemical pharmaceutical industry. In this work, we describe a method of the direct synthesis of amides from carboxylic acids and amines under solvent-free conditions using minute quantities of ceric ammonium nitrate (CAN) as a catalyst. The reactions are carried out in an open microwave reactor and allow the corresponding amides to be obtained in a fast and effective manner when compared to other procedures of the direct synthesis of amides from acids and amines reported so far in the literature. The amide product isolation procedure is simple, environmentally friendly, and is performed with no need for chromatographic purification of secondary amides due to high yields. In this report, primary amines were used in most examples. However, the developed procedure seems to be applicable for secondary amines as well. The methodology produces a limited amount of wastes, and a catalyst can be easily separated. This highly efficient, robust, rapid, solvent-free, and additional reagent-free method provides a major advancement in the development of an ideal green protocol for amide bond formation.

Respiratory syncytial virus co-opts host mitochondrial function to favour infectious virus production.

Although respiratory syncytial virus (RSV) is responsible for more human deaths each year than influenza, its pathogenic mechanisms are poorly understood. Here high-resolution quantitative imaging, bioenergetics measurements and mitochondrial membrane potential- and redox-sensitive dyes are used to define RSV's impact on host mitochondria for the first time, delineating RSV-induced microtubule/dynein-dependent mitochondrial perinuclear clustering, and translocation towards the microtubule-organizing centre. These changes are concomitant with impaired mitochondrial respiration, loss of mitochondrial membrane potential and increased production of mitochondrial reactive oxygen species (ROS). Strikingly, agents that target microtubule integrity the dynein motor protein, or inhibit mitochondrial ROS production strongly suppresses RSV virus production, including in a mouse model with concomitantly reduced virus-induced lung inflammation. The results establish RSV's unique ability to co-opt host cell mitochondria to facilitate viral infection, revealing the RSV-mitochondrial interface for the first time as a viable target for therapeutic intervention.

Evidence for Reagent-Induced Spin-State Switching in Tripodal Fe(II) Iminopyridine Complexes.

We present evidence of a spin-state change that accompanies desilylation reactions performed on two related Fe(II) iminopyridine coordination complexes. To probe these systems, we performed titrations with CsF in solution and analyzed the speciation with in situ magnetometry, electrochemistry, and mass spectrometry techniques. We find that pendant tert-butyldimethylsilyl groups are readily cleaved under these conditions, and the resulting desilylated complexes exhibit overall decreased solution magnetic susceptibility values. Density functional theory and ab initio computations probe the impact of substituent identity (prior to- and post-desilylation) on the metal-ligand σ-donor and π-acceptor bonding properties. We attribute the observed spin-state changes to the decrease in entropy associated with the conformational freedom of the silylated high-spin complex, resulting in a more favored low-spin state upon desilylation.

EU-OPENSCREEN: A Novel Collaborative Approach to Facilitate Chemical Biology.

Compound screening in biological assays and subsequent optimization of hits is indispensable for the development of new molecular research tools and drug candidates. To facilitate such discoveries, the European Research Infrastructure EU-OPENSCREEN was founded recently with the support of its member countries and the European Commission. Its distributed character harnesses complementary knowledge, expertise, and instrumentation in the discipline of chemical biology from 20 European partners, and its open working model ensures that academia and industry can readily access EU-OPENSCREEN's compound collection, equipment, and generated data. To demonstrate the power of this collaborative approach, this perspective article highlights recent projects from EU-OPENSCREEN partner institutions. These studies yielded (1) 2-aminoquinazolin-4(3 H)-ones as potential lead structures for new antimalarial drugs, (2) a novel lipodepsipeptide specifically inducing apoptosis in cells deficient for the pVHL tumor suppressor, (3) small-molecule-based ROCK inhibitors that induce definitive endoderm formation and can potentially be used for regenerative medicine, (4) potential pharmacological chaperones for inborn errors of metabolism and a familiar form of acute myeloid leukemia (AML), and (5) novel tankyrase inhibitors that entered a lead-to-candidate program. Collectively, these findings highlight the benefits of small-molecule screening, the plethora of assay designs, and the close connection between screening and medicinal chemistry within EU-OPENSCREEN.

Drawing on biology to inspire molecular design: a redox-responsive MRI probe based on Gd(iii)-nicotinamide.

A novel, reversible redox-active MRI probe, GdNR1, has been developed for the study of redox changes associated with diseased states. This system exhibits switching in relaxivity upon reduction and oxidation of the appended nicotinimidium. Relaxivity studies and cyclic voltammetry confirmed the impressive reversibility of this system, at a biologically-relevant reduction potential. A 2.5-fold increase in relaxivity was observed upon reduction of the complex, which corresponds to a change in the number of inner-sphere water molecules, as confirmed by luminescence lifetimes of the Eu(iii) analogue and NMRD studies. This is the first example of a redox-responsive MRI probe utilising the biologically-inspired nicotinimidium redox switch. In the future this strategy could enable the non-invasive identification of hypoxic tissue and related cardiovascular disease.

A reversible fluorescent probe for monitoring Ag(I) ions.

Silver-containing nanomaterials are of interest for their antibiotic properties, for a wide range of applications from medicine to consumer products. However, much remains to be learnt about the degradation of such materials and their effects on human health. While most analyses involve measurement of total silver levels, it is important also to be able to measure concentrations of active free Ag(I) ions. We report here the preparation of a coumarin-based probe, thiocoumarin silver sensor 1 (TcAg1), that responds reversibly to the addition of silver ions through the appearance of a new fluorescence emission peak at 565 nm. Importantly, this peak is not observed in the presence of Hg(II), a common interferent in Ag(I) sensing. To establish the utility of this sensor, we prepared silver-doped phosphate glasses with demonstrated bactericidal properties, and observed the Ag(I) release from these glasses in solutions of different ionic strength. TcAg1 is therefore a useful tool for the study of the environmental and medical effects of silver-containing materials.

A ratiometric iron probe enables investigation of iron distribution within tumour spheroids.

Iron dysregulation is implicated in numerous diseases, and iron homeostasis is profoundly influenced by the labile iron pool (LIP). Tools to easily observe changes in the LIP are limited, with calcein AM-based assays most widely used. We describe here FlCFe1, a ratiometric analogue of calcein AM, which also provides the capacity for imaging iron in 3D cell models.

Pro-fluorescent mitochondria-targeted real-time responsive redox probes synthesised from carboxy isoindoline nitroxides: Sensitive probes of mitochondrial redox status in cells.

Here we describe new fluorescent probes based on fluorescein and rhodamine that provide reversible, real-time insight into cellular redox status. The new probes incorporate bio-imaging relevant fluorophores derived from fluorescein and rhodamine linked with stable nitroxide radicals such that they cannot be cleaved, either spontaneously or enzymatically by cellular processes. Overall fluorescence emission is determined by reversible reduction and oxidation, hence the steady state emission intensity reflects the balance between redox potentials of critical redox couples within the cell. The permanent positive charge on the rhodamine-based probes leads to their rapid localisation within mitochondria in cells. Reduction and oxidation also leads to marked changes in the fluorophore lifetime, enabling monitoring by fluorescence lifetime imaging microscopy. Finally, we demonstrate that administration of a methyl ester version of the rhodamine-based probe can be used at concentrations as low as 5 nM to generate a readily detected response to redox stress within cells as analysed by flow cytometry.

Fluorescent probes for the simultaneous detection of multiple analytes in biology.

Many of the key questions facing cellular biology concern the location and concentration of chemical species, from signalling molecules to metabolites to exogenous toxins. Fluorescent sensors (probes) have revolutionised the understanding of biological systems through their exquisite sensitivity to specific analytes. Probe design has focussed on selective sensors for individual analytes, but many of the most pertinent biological questions are related to the interaction of more than one chemical species. While it is possible to simultaneously use multiple sensors for such applications, data interpretation will be confounded by the fact that sensors will have different uptake, localisation and metabolism profiles. An alternative solution is to instead use a single probe that responds to two analytes, termed a dual-responsive probe. Recent progress in this field has yielded exciting probes, some of which have demonstrated biological application. Here we review work that has been carried out to date, and suggest future research directions that will harness the considerable potential of dual-responsive fluorescent probes.

A Carborane-Containing Fluorophore as a Stain of Cellular Lipid Droplets.

The use of fluorescent markers and probes greatly enhances biological investigations but relies on the provision of an array of fluorophores with diverse properties. Herein we report a novel carborane-containing coumarin, 5, which is sufficiently lipophilic to localise in cellular lipid droplets. In non-polar solvents which show comparable polarities to those of a lipid environment, compound 5 exhibits a fluorescence quantum yield two orders of magnitude greater than found in aqueous solvents, adding a further degree of selectivity to lipid droplet imaging. Compound 5 can stain lipid droplets in ex vivo adipocytes as well as in cultured cells, and can be utilised in flow cytometry as well as confocal microscopy.

Mitochondrially targeted fluorescent redox sensors.

The balance of oxidants and antioxidants within the cell is crucial for maintaining health, and regulating physiological processes such as signalling. Consequently, imbalances between oxidants and antioxidants are now understood to lead to oxidative stress, a physiological feature that underlies many diseases. These processes have spurred the field of chemical biology to develop a plethora of sensors, both small-molecule and fluorescent protein-based, for the detection of specific oxidizing species and general redox balances within cells. The mitochondrion, in particular, is the site of many vital redox reactions. There is therefore a need to target redox sensors to this particular organelle. It has been well established that targeting mitochondria can be achieved by the use of a lipophilic cation-targeting group, or by utilizing natural peptidic mitochondrial localization sequences. Here, we review how these two approaches have been used by a number of researchers to develop mitochondrially localized fluorescent redox sensors that are already proving useful in providing insights into the roles of reactive oxygen species in the mitochondria.

A ratiometric fluorescent sensor for the mitochondrial copper pool.

Copper plays a key role in the modulation of cellular function, defence, and growth. Here we present InCCu1, a ratiometric fluorescent sensor for mitochondrial copper, which changes from red to blue emission in the presence of Cu(i). Employing this probe in microscopy and flow cytometry, we show that cisplatin-treated cells have an impaired ability to accumulate copper in the mitochondria.

Selective and Reversible Approaches Toward Imaging Redox Signaling Using Small-Molecule Probes.

SIGNIFICANCE: Recent research has identified key roles for reactive oxygen species (ROS)/reactive nitrogen species (RNS) in redox signaling, but much remains to be uncovered. Molecular imaging tools to study these processes must not only be selective to enable identification of the ROS/RNS involved but also reversible to distinguish signaling processes from oxidative stress. Fluorescent sensors offer the potential to image such processes with high spatial and temporal resolution. RECENT ADVANCES: A broad array of strategies has been developed that enable the selective sensing of ROS/RNS. More recently, attention has turned to the design of reversible small-molecule sensors of global redox state, with a further set of probes capable of reversible sensing of individual ROS/RNS. CRITICAL ISSUES: In this study, we discuss the key challenges in achieving simultaneous detection of reversible oxidative bursts with unambiguous determination of a particular ROS/RNS. FUTURE DIRECTIONS: We have highlighted key design features of small-molecule probes that show promise in enabling the study of redox signaling, identifying essential parameters that must be assessed for any new probe. Antioxid. Redox Signal. 24, 713-730.

Reversible Fluorescent Probes for Biological Redox States.

The redox chemistry of the cell is key to its function and health, and the development of chemical tools to study redox biology is important. While fluxes in oxidative state are essential for healthy cell function, a chronically elevated oxidative capacity is linked to disease. It is therefore essential that probes of biological redox states distinguish between these two conditions by the reversible sensing of changes over time. In this review, we discuss the current progress towards such probes, and identify key directions for future research in this nascent field of vital biological interest.