Health-related quality of life after restrictive versus liberal RBC transfusion for cardiac surgery: Sub-study from a randomized clinical trial.

BACKGROUND: Transfusion Requirements in Cardiac Surgery III (TRICS III), a multi-center randomized controlled trial, demonstrated clinical non-inferiority for restrictive versus liberal RBC transfusion for patients undergoing cardiac surgery. However, it is uncertain if transfusion strategy affects long-term health-related quality of life (HRQOL). STUDY DESIGN AND METHODS: In this planned sub-study of Australian patients in TRICS III, we sought to determine the non-inferiority of restrictive versus liberal transfusion strategy on long-term HRQOL and to describe clinical outcomes 24 months postoperatively. The restrictive strategy involved transfusing RBCs when hemoglobin was <7.5 g/dl; the transfusion triggers in the liberal group were: <9.5 g/L intraoperatively, <9.5 g/L in intensive care, or <8.5 g/dl on the ward. HRQOL assessments were performed using the 36-item short form survey version 2 (SF-36v2). Primary outcome was non-inferiority of summary measures of SF-36v2 at 12 months, (non-inferiority margin: -0.25 effect size; restrictive minus liberal scores). Secondary outcomes included non-inferiority of HRQOL at 18 and 24 months. RESULTS: Six hundred seventeen Australian patients received allocated randomization; HRQOL data were available for 208/311 in restrictive and 217/306 in liberal group. After multiple imputation, non-inferiority of restrictive transfusion at 12 months was not demonstrated for HRQOL, and the estimates were directionally in favor of liberal transfusion. Non-inferiority also could not be concluded at 18 and 24 months. Sensitivity analyses supported these results. There were no differences in quality-adjusted life years or composite clinical outcomes up to 24 months after surgery. DISCUSSION: The non-inferiority of a restrictive compared to a liberal transfusion strategy was not established for long-term HRQOL in this dataset.

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.

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.

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.

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.

Coding and long non-coding RNAs provide evidence of distinct transcriptional reprogramming for two ecotypes of the extremophile plant Eutrema salsugineum undergoing water deficit stress.

BACKGROUND: The severity and frequency of drought has increased around the globe, creating challenges in ensuring food security for a growing world population. As a consequence, improving water use efficiency by crops has become an important objective for crop improvement. Some wild crop relatives have adapted to extreme osmotic stresses and can provide valuable insights into traits and genetic signatures that can guide efforts to improve crop tolerance to water deficits. Eutrema salsugineum, a close relative of many cruciferous crops, is a halophytic plant and extremophyte model for abiotic stress research. RESULTS: Using comparative transcriptomics, we show that two E. salsugineum ecotypes display significantly different transcriptional responses towards a two-stage drought treatment. Even before visibly wilting, water deficit led to the differential expression of almost 1,100 genes for an ecotype from the semi-arid, sub-arctic Yukon, Canada, but only 63 genes for an ecotype from the semi-tropical, monsoonal, Shandong, China. After recovery and a second drought treatment, about 5,000 differentially expressed genes were detected in Shandong plants versus 1,900 genes in Yukon plants. Only 13 genes displayed similar drought-responsive patterns for both ecotypes. We detected 1,007 long non-protein coding RNAs (lncRNAs), 8% were only expressed in stress-treated plants, a surprising outcome given the documented association between lncRNA expression and stress. Co-expression network analysis of the transcriptomes identified eight gene clusters where at least half of the genes in each cluster were differentially expressed. While many gene clusters were correlated to drought treatments, only a single cluster significantly correlated to drought exposure in both ecotypes. CONCLUSION: Extensive, ecotype-specific transcriptional reprogramming with drought was unexpected given that both ecotypes are adapted to saline habitats providing persistent exposure to osmotic stress. This ecotype-specific response would have escaped notice had we used a single exposure to water deficit. Finally, the apparent capacity to improve tolerance and growth after a drought episode represents an important adaptive trait for a plant that thrives under semi-arid Yukon conditions, and may be similarly advantageous for crop species experiencing stresses attributed to climate change.

Repeated presentation of children and adolescents to the emergency department following self-harm: A retrospective audit of hospital data.

OBJECTIVE: To examine re-presentation rates for self-harm in patients aged 0-18 years to the ED of a tertiary paediatric hospital in Melbourne, Australia, and associated patient, family and hospital presentation factors. METHODS: Data for presentations from 1 July 2016 to 31 December 2018 were extracted from the hospital's electronic medical record system. Self-harm presentations were identified through automated, rule-based coding and manual review of medical notes. Re-presentation rates for intervals up to 12 months were estimated using survival methods with risk factor associations examined using Cox regression. RESULTS: Of the 952 presentations for self-harm after 1 January 2017, 529 were considered first presentations. An estimated 15% (95% confidence interval [CI] 12-19), 20% (95% CI 17-24) and 23% (95% CI 19-27) re-presented for self-harm within 3, 6 and 12 months, respectively. A total of 82% of all presentations were for girls. Patients were more likely to re-present if they had previously presented more than once, were flagged as vulnerable (hazard ratio [HR] 1.35, 95% CI 1.08-1.68), had a history of substance abuse (HR 1.30, 95% CI 1.03-1.64), were female (HR 1.43, 95% CI 0.92-2.21), had self-cut (HR 1.38, 95% CI 0.96-1.97), had an aggressive behaviour response team called during the visit (HR 1.44, 95% CI 0.85-2.45) or had a history of depression (HR 1.27, 95% CI 0.99-1.63). CONCLUSIONS: In this paediatric ED, almost one in four patients re-presented with self-harm within 12 months. Previous presentations and other factors were associated with risk of re-presenting, although no factor was strongly predictive. Future research might examine the generalisability of these findings across settings and explore strategies for prevention.

Evidence that tolerance of Eutrema salsugineum to low phosphate conditions is hard-wired by constitutive metabolic and root-associated adaptations.

MAIN CONCLUSION: The extremophyte Eutrema salsugineum (Yukon ecotype) has adapted to an environment low in available phosphate through metabolic and root-associated traits that enables it to efficiently retrieve, use, and recycle phosphorus. Efficient phosphate (Pi) use by plants would increase crop productivity under Pi-limiting conditions and reduce our reliance on Pi applied as fertilizer. An ecotype of Eutrema salsugineum originating from the Yukon, Canada, shows no evidence of decreased relative growth rate or biomass under low Pi conditions and, as such, offers a promising model for identifying mechanisms to improve Pi use by crops. We evaluated traits associated with efficient Pi use by Eutrema (Yukon ecotype) seedlings and 4-week-old plants, including acquisition, remobilization, and the operation of metabolic bypasses. Relative to Arabidopsis, Eutrema was slower to remobilize phosphorus (P) from senescing leaves, primary and lateral roots showed a lower capacity for rhizosphere acidification, and root acid phosphatase activity was more broadly distributed and not Pi responsive. Both species produced long root hairs on low Pi media, whereas Arabidopsis root hairs were well endowed with phosphatase activity. This capacity was largely absent in Eutrema. In contrast to Arabidopsis, maximal in vitro rates of pyrophosphate-dependent phosphofructokinase and phosphoenolpyruvate carboxylase activities were not responsive to low Pi conditions suggesting that Eutrema has a constitutive and likely preferential capacity to use glycolytic bypass enzymes. Rhizosphere acidification, exudation of acid phosphatases, and rapid remobilization of leaf P are unlikely strategies used by Eutrema for coping with low Pi. Rather, equipping an entire root system for Pi acquisition and utilizing a metabolic strategy suited to deficient Pi conditions offer better explanations for how Eutrema has adapted to thrive on alkaline, highly saline soil that is naturally low in available Pi.

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.

Acclimation of the crucifer Eutrema salsugineum to phosphate limitation is associated with constitutively high expression of phosphate-starvation genes.

Eutrema salsugineum, a halophytic relative of Arabidopsis thaliana, was subjected to varying phosphate (Pi) treatments. Arabidopsis seedlings grown on 0.05 mm Pi displayed shortened primary roots, higher lateral root density and reduced shoot biomass allocation relative to those on 0.5 mm Pi, whereas Eutrema seedlings showed no difference in lateral root density and shoot biomass allocation. While a low Fe concentration mitigated the Pi deficiency response for Arabidopsis, Eutrema root architecture was unaltered, but adding NaCl increased Eutrema lateral root density almost 2-fold. Eutrema and Arabidopsis plants grown on soil without added Pi for 4 weeks had low shoot and root Pi content. Pi-deprived, soil-grown Arabidopsis plants were stunted with senescing older leaves, whereas Eutrema plants were visually indistinguishable from 2.5 mm Pi-supplemented plants. Genes associated with Pi starvation were analysed by RT-qPCR. EsIPS2, EsPHT1;4 and EsPAP17 showed up-regulated expression in Pi-deprived Eutrema plants, while EsPHR1, EsWRKY75 and EsRNS1 showed no induction. Absolute quantification of transcripts indicated that PHR1, WRKY75 and RNS1 were expressed at higher levels in Eutrema plants relative to those in Arabidopsis regardless of external Pi. The low phenotypic plasticity Eutrema displays to Pi supply is consistent with adaptation to chronic Pi deprivation in its extreme natural habitat.