Cationic helicenes as selective G4 DNA binders and optical probes for cellular imaging.

The important role that G-quadruplex DNA (G4 DNA) structures play in regulating biological processes is becoming widely recognised. These structures have also been proposed to be attractive drug targets. Therefore, there has been significant interest in developing small molecules that can selectively bind to G4 DNA over other topologies. In this paper we investigate the interaction between DNA and helical compounds (helicenes) based on a central carbocation trisubstituted with aromatic rings. We show that the non-planar structure of these helicenes results in a significantly reduced affinity for dsDNA when compared to their planar analogues, whilst maintaining a high affinity for G4 DNA. Additionally, the right- and left-handed enantiomers of one of these helicenes recognise the chiral DNA environments of G4 and dsDNA differently. We show that upon DNA binding the helicenes display a fluorescence switch-on effect, which we have successfully used for cellular imaging in live and fixed U2OS cells, staining mitochondria and the nucleus, respectively.

Selective Detection of Cu+ Ions in Live Cells via Fluorescence Lifetime Imaging Microscopy.

Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).

Rotaxanes as Cages to Control DNA Binding, Cytotoxicity, and Cellular Uptake of a Small Molecule*.

The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and "off-target" interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to "cage" a DNA-targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G-quadruplex DNA binder, namely a PtII -salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII -salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII -salphen to the nucleus and its binding to DNA.

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy.

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

Assessing The Key Photophysical Properties of Triangulenium Dyes for DNA Binding by Alteration of the Fluorescent Core.

Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.

Design, synthesis and evaluation of a tripodal receptor for phosphatidylinositol phosphates.

Phosphatidylinositol phosphates (PIPs) are membrane phospholipids that play crucial roles in a wide range of cellular processes. Their function is dictated by the number and positions of the phosphate groups in the inositol ring (with seven different PIPs being active in the cell). Therefore, there is significant interest in developing small-molecule receptors that can bind selectively to these species and in doing so affect their cellular function or be the basis for molecular probes. However, to date there are very few examples of such molecular receptors. Towards this aim, herein we report a novel tripodal molecule that acts as receptor for mono- and bis-phosphorylated PIPs in a cell free environment. To assess their affinity to PIPs we have developed a new cell free assay based on the ability of the receptor to prevent alkaline phosphatase from hydrolysing these substrates. The new receptor displays selectivity towards two out of the seven PIPs, namely PI(3)P and PI(3,4)P2. To rationalise these results, a DFT computational study was performed which corroborated the experimental results and provided insight into the host-guest binding mode.

Transcutaneous fluorescence spectroscopy as a tool for non-invasive monitoring of gut function: first clinical experiences.

Gastro-intestinal function plays a vital role in conditions ranging from inflammatory bowel disease and HIV through to sepsis and malnutrition. However, the techniques that are currently used to assess gut function are either highly invasive or unreliable. Here we present an alternative, non-invasive sensing modality for assessment of gut function based on fluorescence spectroscopy. In this approach, patients receive an oral dose of a fluorescent contrast agent and a fibre-optic probe is used to make fluorescence measurements through the skin. This provides a readout of the degree to which fluorescent dyes have permeated from the gut into the blood stream. We present preliminary results from our first measurements in human volunteers demonstrating the potential of the technique for non-invasive monitoring of multiple aspects of gastro-intestinal health.

Unravelling the Enzymatic Degradation Mechanism of Supramolecular Peptide Nanofibers and Its Correlation with Their Internal Viscosity.

Enzyme-responsive supramolecular peptide biomaterials have attracted growing interest for disease diagnostics and treatments. However, it remains unclear whether enzymes target the peptide assemblies or dissociated peptide monomers. To gain further insight into the degradation mechanism of supramolecular peptide amphiphile (PA) nanofibers, cathepsin B with both exopeptidase and endopeptidase activities was exploited here for degradation studies. Hydrolysis was found to occur directly on the PA nanofibers as only surface amino acid residues were cleaved. The number of cleaved residues and the degradation efficiency was observed to be negatively correlated with the internal viscosity of the PA nanofibers, quantified to be between 200-800 cP (liquid phase) using fluorescence lifetime imaging microscopy combined with an environmentally sensitive molecular rotor, BODIPY-C10. These findings enhance our understanding on the enzymatic degradation of supramolecular PA nanofibers and have important implications for the development of PA probes for the real-time monitoring of disease-related enzymes.

Rapid formation of human immunodeficiency virus-like particles.

Understanding the molecular mechanisms involved in the assembly of viruses is essential for discerning how viruses transmit from cell to cell and host to host. Although molecular aspects of assembly have been studied for many viruses, we still have little information about these events in real time. Enveloped viruses such as HIV that assemble at, and bud from, the plasma membrane have been studied in some detail using live cell fluorescence imaging techniques; however, these approaches provide little information about the real-time morphological changes that take place as viral components come together to form individual virus particles. Here we used correlative scanning ion conductance microscopy and fluorescence confocal microscopy to measure the topological changes, together with the recruitment of fluorescently labeled viral proteins such as Gag and Vpr, during the assembly and release of individual HIV virus-like particles (VLPs) from the top, nonadherent surfaces of living cells. We show that 1) labeling of viral proteins with green fluorescent protein affects particle formation, 2) the kinetics of particle assembly on different plasma membrane domains can vary, possibly as a consequence of differences in membrane biophysical properties, and 3) VLPs budding from the top, unimpeded surface of cells can reach full size in 20 s and disappear from the budding site in 0.5 to 3 min from the moment curvature is initially detected, significantly faster than has been previously reported.

Microscopic Viscosity of Neuronal Plasma Membranes Measured Using Fluorescent Molecular Rotors: Effects of Oxidative Stress and Neuroprotection.

Molecular mobility in neuronal plasma membranes is a crucial factor in brain function. Microscopic viscosity is an important parameter that determines molecular mobility. This study presents the first direct measurement of the microviscosity of plasma membranes of live neurons. Microviscosity maps were obtained using fluorescence lifetime imaging of environment-sensing dyes termed "molecular rotors". Neurons were investigated both in the basal state and following common neurodegenerative stimuli, excitotoxicity, or oxidative stress. Both types of neurotoxic challenges induced microviscosity decrease in cultured neurons, and oxidant-induced membrane fluidification was counteracted by the wide-spectrum neuroprotectant, the H3 peptide. These results provide new insights into molecular mobility in neuronal membranes, paramount for basic brain function, and suggest that preservation of membrane stability may be an important aspect of neuroprotection in brain insults and neurodegenerative disorders.

Internet of things and Big Data as potential solutions to the problems in waste electrical and electronic equipment management: An exploratory study.

Management of Waste Electrical and Electronic Equipment (WEEE) is a vital part in solid waste management, there are still some difficult issues require attentionss. This paper investigates the potential of applying Internet of Things (IoT) and Big Data as the solutions to the WEEE management problems. The massive data generated during the production, consumption and disposal of Electrical and Electronic Equipment (EEE) fits the characteristics of Big Data. Through using the state-of-the-art communication technologies, the IoT derives the WEEE "Big Data" from the life cycle of EEE, and the Big Data technologies process the WEEE "Big Data" for supporting decision making in WEEE management. The framework of implementing the IoT and the Big Data technologies is proposed, with its multiple layers are illustrated. Case studies with the potential application scenarios of the framework are presented and discussed. As an unprecedented exploration, the combined application of the IoT and the Big Data technologies in WEEE management brings a series of opportunities as well as new challenges. This study provides insights and visions for stakeholders in solving the WEEE management problems under the context of IoT and Big Data.

From waste plastics to industrial raw materials: A life cycle assessment of mechanical plastic recycling practice based on a real-world case study.

Mechanical recycling of waste plastics is an environmental solution to the problem of waste plastic disposal, and has already become a common practice in industry. However, limited information can be found on either the industralised plastic recycling or the recycled materials, despite the use of recycled plastics has already extended to automobile production. This study investigates the life cycle environmental impacts of mechanical plastic recycling practice of a plastic recycling company in China. Waste plastics from various sources, such as agricultural wastes, plastic product manufacturers, collected solid plastic wastes and parts dismantled from waste electric and electronic equipments, are processed in three routes with products end up in different markets. The results of life cycle assessments show that the extrusion process has the largest environmental impacts, followed by the use of fillers and additives. Compared to production of virgin plastics and composites, the mechanical recycling is proved to be a superior alternative in most environmental aspects. Substituting virgin plastic composites with recycled plastic composites has achieved the highest environmental benefits, as virgin composite production has an impact almost 4 times higher that of the recycled composite production in each ReCiPe endpoint damage factor. Sensitivity analysis shows that the coverage of collecting network contribute affect little to overall environmental impact, and centralisation plays an important role in reducing overall environmental impacts. Among the fillers and additives, impact modifiers account for the most significant contributions to the environmental impacts of recycled composites. This study provides necessary information about the existing industrialised plastic recycling practice, and recommendations are given. Research implications are presented with the purpose to achieve higher substitution rate and lower environmental impact.

Synthesis and Photophysical Study of a [NiFe] Hydrogenase Biomimetic Compound Covalently Linked to a Re-diimine Photosensitizer.

The synthesis, photophysics, and photochemistry of a linked dyad ([Re]-[NiFe2]) containing an analogue ([NiFe2]) of the active site of [NiFe] hydrogenase, covalently bound to a Re-diimine photosensitizer ([Re]), are described. Following excitation, the mechanisms of electron transfer involving the [Re] and [NiFe2] centers and the resulting decomposition were investigated. Excitation of the [Re] center results in the population of a diimine-based metal-to-ligand charge transfer excited state. Reductive quenching by NEt3 produces the radically reduced form of [Re], [Re](-) (kq = 1.4 ± 0.1 × 10(7) M(-1) s(-1)). Once formed, [Re](-) reduces the [NiFe2] center to [NiFe2](-), and this reduction was followed using time-resolved infrared spectroscopy. The concentration dependence of the electron transfer rate constants suggests that both inter- and intramolecular electron transfer pathways are involved, and the rate constants for these processes have been estimated (kinter = 5.9 ± 0.7 × 10(8) M(-1) s(-1), kintra = 1.5 ± 0.1 × 10(5) s(-1)). For the analogous bimolecular system, only intermolecular electron transfer could be observed (kinter = 3.8 ± 0.5 × 10(9) M(-1) s(-1)). Fourier transform infrared spectroscopic studies confirms that decomposition of the dyad occurs upon prolonged photolysis, and this appears to be a major factor for the low activity of the system toward H2 production in acidic conditions.

Comparison of rhenium-porphyrin dyads for CO2 photoreduction: photocatalytic studies and charge separation dynamics studied by time-resolved IR spectroscopy.

We report a study of the photocatalytic reduction of CO2 to CO by zinc porphyrins covalently linked to [ReI(2,2'-bipyridine)(CO)3L]+/0 moieties with visible light of wavelength >520 nm. Dyad 1 contains an amide C6H4NHC(O) link from porphyrin to bipyridine (Bpy), Dyad 2 contains an additional methoxybenzamide within the bridge C6H4NHC(O)C6H3(OMe)NHC(O), while Dyad 3 has a saturated bridge C6H4NHC(O)CH2; each dyad is studied with either L = Br or 3-picoline. The syntheses, spectroscopic characterisation and cyclic voltammetry of Dyad 3 Br and [Dyad 3 pic]OTf are described. The photocatalytic performance of [Dyad 3 pic]OTf in DMF/triethanolamine (5 : 1) is approximately an order of magnitude better than [Dyad 1 pic]PF6 or [Dyad 2 pic]OTf in turnover frequency and turnover number, reaching a turnover number of 360. The performance of the dyads with Re-Br units is very similar to that of the dyads with [Re-pic]+ units in spite of the adverse free energy of electron transfer. The dyads undergo reactions during photocatalysis: hydrogenation of the porphyrin to form chlorin and isobacteriochlorin units is detected by visible absorption spectroscopy, while IR spectroscopy reveals replacement of the axial ligand by a triethanolaminato group and insertion of CO2 into the latter to form a carbonate. Time-resolved IR spectra of [Dyad 2 pic]OTf and [Dyad 3 pic]OTf (560 nm excitation in CH2Cl2) demonstrated electron transfer from porphyrin to Re(Bpy) units resulting in a shift of ν(CO) bands to low wavenumbers. The rise time of the charge-separated species for [Dyad 3 pic]OTf is longest at 8 (±1) ps and its lifetime is also the longest at 320 (±15) ps. The TRIR spectra of Dyad 1 Br and Dyad 2 Br are quite different showing a mixture of 3MLCT, IL and charge-separated excited states. In the case of Dyad 3 Br, the charge-separated state is absent altogether. The TRIR spectra emphasize the very different excited states of the bromide complexes and the picoline complexes. Thus, the similarity of the photocatalytic data for bromide and picoline dyads suggests that they share common intermediates. Most likely, these involve hydrogenation of the porphyrin and substitution of the axial ligand at rhenium.

Photochemical dihydrogen production using an analogue of the active site of [NiFe] hydrogenase.

Photoproduction of dihydrogen (H2) by a low molecular weight analogue of the active site of [NiFe] hydrogenase has been investigated by reduction of the [NiFe2] cluster, 1, by a photosensitier PS (PS = [ReCl(CO)3(bpy)] or [Ru(bpy)3][PF6]2). Reductive quenching of the (3)MLCT excited state of the photosensitizer by NEt3 or N(CH2CH2OH)3 (TEOA) generates PS(•-), and subsequent intermolecular electron transfer to 1 produces the reduced anionic form of 1. Time-resolved infrared spectroscopy (TRIR) has been used to probe the intermediates throughout the reduction of 1 and subsequent photocatalytic H2 production from [HTEOA][BF4], which was monitored by gas chromatography. Two structural isomers of the reduced form of 1 (1a(•-) and 1b(•-)) were detected by Fourier transform infrared spectroscopy (FTIR) in both CH3CN and DMF (dimethylformamide), while only 1a(•-) was detected in CH2Cl2. Structures for these intermediates are proposed from the results of density functional theory calculations and FTIR spectroscopy. 1a(•-) is assigned to a similar structure to 1 with six terminal carbonyl ligands, while calculations suggest that in 1b(•-) two of the carbonyl groups bridge the Fe centers, consistent with the peak observed at 1714 cm(-1) in the FTIR spectrum for 1b(•-) in CH3CN, assigned to a ν(CO) stretching vibration. Formation of 1a(•-) and 1b(•-) and production of H2 was studied in CH3CN, DMF, and CH2Cl2. Although the more catalytically active species (1a(•-) or 1b(•-)) could not be determined, photocatalysis was observed only in CH3CN and DMF.