The Mannich reaction of hydrazones: improved reactivity under solvent-free conditions

The Mannich reaction of hydrazones can be performed without solvent by simply mixing the hydrazone with two to three equivalents of a secondary amine and formaldehyde. Best yields and conditions are obtained with p-nitro substituted arylhydrazones. These conditions allow the efficient coupling of simple hydrazones that failed to react in toluene or ethanol solutions.

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions.

The effects of retinoic acid on human corneal stromal keratocytes cultured in vitro under serum-free conditions

Purpose: Retinoic acid (RA) is a metabolite of vitamin A that plays a fundamental role in the development and function of the human eye. The purpose of this study was to investigate the effects of RA on the phenotype of corneal stromal keratocytes maintained in vitro for extended periods under serum-free conditions. Methods: Keratocytes isolated from human corneas were cultured up to 21 days in serum-free media supplemented with RA or DMSO vehicle. The effects of RA and of its removal after treatment on cell proliferation and morphology were evaluated. In addition, the expression of keratocyte markers was quantified at the transcriptional and protein levels by quantitative PCR and immunoblotting or ELISA, respectively. Furthermore, the effects of RA on keratocyte migration were tested using scratch assays. Results: Keratocytes cultured with RA up to 10×10-6 M showed enhanced proliferation and stratification, and reduced mobility. RA also promoted the expression of keratocyte-characteristic proteoglycans such as keratocan, lumican, and decorin, and increased the amounts of collagen type-I in culture while significantly reducing the expression of matrix metalloproteases 1, 3, and 9. RA effects were reversible, and cell phenotype reverted to that of control after removal of RA from media. Conclusions: RA was shown to control the phenotype of human corneal keratocytes cultured in vitro by regulating cell behaviour and extracellular matrix composition. These findings contribute to our understanding of corneal stromal biology in health and disease, and may prove useful in optimizing keratocyte cultures for applications in tissue engineering, cell biology, and medicine.

Lightly branched comb polyesters: application in fast drying solvent-borne coating formulations

Novel oxazoline-based comb-polymers possessing linoleyl or oleic side chains have been synthesized and used to produce low viscosity coatings. Inclusion of the polymers in model paint formulations results in coatings that exhibit faster drying times than commercially available alkyd resin formulations. The comb polymers were produced from diol substituted oxazoline monomers that were synthesized through a scalable, solvent free protocol and purified by simple recrystallisation. Co-polymerisation of the oxazolines with adipic acid at 160 °C in the bulk resulted in the targeted polyester comb type polymers. The polymers were soluble in a range of organic solvents and compatible with commercial alkyd resins. Model paint formulations containing up to 40 wt% of the linoleyl-based comb polymers exhibited a dramatic reduction in viscosity (from 35 to 13 Poise at 25 °C) with increasing quantities of polymer added. Dynamic mechanical analysis (DMA) studies revealed that the drying rate of the model paint formulations containing the comb polymers was enhanced when compared with that of commercial alkyd resins.

Mechanisms for the formation of organic acids in the gas-phase ozonolysis of 3-carene

This paper describes experimental studies aimed at elucidating mechanisms for the formation of low-volatility organic acids in the gas-phase ozonolysis of 3-carene. Experiments were carried out in a static chamber under 'OH-free' conditions. A range of multifunctional acids-which are analogous to those observed from alpha-pinene ozonolysis-were identified in the condensed phase using gas chromatography coupled to mass spectrometry after derivation. Product yields were determined as a function of different OH radical scavengers and relative humidities to give mechanistic information about their routes of formation. Furthermore, an enone and an enal derived from 3-carene were ozonised in order to probe the early mechanistic steps in the reaction and, in particular, which of the two initially formed Criegee intermediates gives rise to which products. Branching ratios for the formation of the two Criegee Intermediates are determined. Similarities and differences in product formation from 3-carene and alpha-pinene ozonolysis are discussed and possible mechanisms-supported by experimental evidence-are developed for all acids investigated.

Mechanisms for the formation of secondary organic aerosol components from the gas-phase ozonolysis of alpha-pinene

Gas-phase ozonolysis of alpha-pinene was studied in static chamber experiments under 'OH-free' conditions. A range of multifunctional products-in particular low-volatility carboxylic acids-were identified in the condensed phase using gas chromatography coupled to mass spectrometry after derivatisation. The dependence of product yields on reaction conditions (humidity, choice of OH radical scavengers, added Criegee intermediate scavengers, NO2 etc.) was investigated to probe the mechanisms of formation of these products; additional information was obtained by studying the ozonolysis of an enal and an enone derived from alpha-pinene. On the basis of experimental findings, previously suggested mechanisms were evaluated and detailed gas-phase mechanisms were developed to explain the observed product formation. Atmospheric implications of this work are discussed.

The influence of ultraviolet radiation on growth, photosynthesis and phenolic levels of green and red lettuce: potential for exploiting effects of ultraviolet radiation in a production system

Studies have shown that natural ultraviolet (UV) radiation increases secondary products such as phenolics but can significantly inhibit biomass accumulation in lettuce plants. In the work presented here, the effect of UV radiation on phenolic concentration and biomass accumulation was assessed in relation to photosynthetic performance in red and green lettuce types. Lettuce plants in polythene clad tunnels were exposed to either ambient (UV transparent film) or UV-free conditions (UV blocking film). The study tested whether growth reduction in lettuce plants exposed to natural UV radiation is because of inhibition of photosynthesis by direct damage to the photosynthetic apparatus or by internal shading by anthocyanins. Ambient levels of UV radiation did not limit the efficiency of photosynthesis suggesting that phenolic compounds may effectively protect the photosynthetic apparatus. Growth inhibition does, however, occur in red lettuce and could be explained by the high metabolic cost of phenolic compounds for UV protection. From a commercial perspective, UV transparent and UV blocking films offer opportunities because, in combination, they could increase plant quality as well as productivity. Growing plants continuously under a UV blocking film, and then 6 days before the final harvest transferring them to a UV transparent film, showed that high yields and high phytochemical content can be achieved complementarily.

Structure variation and evolution in microphase-separated grafted diblock copolymer films

The phase behavior of grafted d-polystyrene-block-poly(methyl methacrylate) diblock copolymer films is examined, with particular focus on the effect of solvent and annealing time. It was observed that the films undergo a two-step transformation from an initially disordered state, through an ordered metastable state, to the final equilibrium configuration. It was also found that altering the solvent used to wash the films, or complete removal of the solvent prior to thermal annealing using supercritical CO2, could influence the structure of the films in the metastable state, though the final equilibrium state was unaffected. To aid in the understanding to these experimental results, a series of self-consistent field theory calculations were done on a model diblock copolymer brush containing solvent. Of the different models examined, those which contained a solvent selective for the grafted polymer block most accurately matched the observed experimental behavior. We hypothesize that the structure of the films in the metastable state results from solvent enrichment of the film near the film/substrate interface in the case of films washed with solvent or faster relaxation of the nongrafted block for supercritical CO2 treated (solvent free) films. The persistence of the metastable structures was attributed to the slow reorganization of the polymer chains in the absence of solvent.

Eigenstructure assignment in descriptor systems

Coordinate free conditions are given for pole assignment by feedback in linear descriptor (singular) systems which guarantee closed-loop regularity. These conditions are shown to be both necessary and sufficient for assignment of the maximum possible number of finite poles. Transformation to special coordinates are not used and the results provide a robust algorithm for the computation of the required feedback.

The roles of aerosol, water vapor and cloud in future global dimming/brightening

Observational evidence indicates significant regional trends in solar radiation at the surface in both all-sky and cloud-free conditions. Negative trends in the downwelling solar surface irradiance (SSI) have become known as ‘dimming’ while positive trends have become known as ‘brightening’. We use the Met Office Hadley Centre HadGEM2 climate model to model trends in cloud-free and total SSI from the pre-industrial to the present-day and compare these against observations. Simulations driven by CMIP5 emissions are used to model the future trends in dimming/brightening up to the year 2100. The modeled trends are reasonably consistent with observed regional trends in dimming and brightening which are due to changes in concentrations in anthropogenic aerosols and, potentially, changes in cloud cover owing to the aerosol indirect effects and/or cloud feedback mechanisms. The future dimming/brightening in cloud-free SSI is not only caused by changes in anthropogenic aerosols: aerosol impacts are overwhelmed by a large dimming caused by increases in water vapor. There is little trend in the total SSI as cloud cover decreases in the climate model used here, and compensates the effect of the change in water vapor. In terms of the surface energy balance, these trends in SSI are obviously more than compensated by the increase in the downwelling terrestrial irradiance from increased water vapor concentrations. However, the study shows that while water vapor is widely appreciated as a greenhouse gas, water vapor impacts on the atmospheric transmission of solar radiation and the future of global dimming/brightening should not be overlooked.

Cannabidivarin is anticonvulsant in mouse and rat in vitro and in seizure models

Summary Background and purpose: Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models. Experimental approach: The effect of CBDV (1-100μM) on epileptiform local field potentials (LFPs) induced in rat hippocampal brain slices by 4-AP application or Mg2+-free conditions was assessed by in vitro multi-electrode array recordings. Additionally, the anticonvulsant profile of CBDV (50-200 mg kg-1) in vivo was investigated in four rodent seizure models: maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures in rat. CBDV effects in combination with commonly-used antiepileptic drugs were investigated in rat seizures. Finally, the motor side effect profile of CBDV was investigated using static beam and gripstrength assays. Key results: CDBV significantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg2+-free conditions. CBDV had significant anticonvulsant effects in mES (≥100 mg kg-1), audiogenic (≥50 mg kg-1) and PTZ-induced seizures (≥100 mg kg-1). CBDV alone had no effect against pilocarpine-induced seizures, but significantly attenuated these seizures when administered with valproate or phenobarbital at 200 mg kg-1 CBDV. CBDV had no effect on motor function. Conclusions and Implications: These results indicate that CBDV is an effective anticonvulsant across a broad range of seizure models, does not significantly affect normal motor function and therefore merits further investigation in chronic epilepsy models to justify human trials.

The role of oceans and sea ice in abrupt transitions between multiple climate states

The coupled climate dynamics underlying large, rapid, and potentially irreversible changes in ice cover are studied. A global atmosphere–ocean–sea ice general circulation model with idealized aquaplanet geometry is forced by gradual multi-millennial variations in solar luminosity. The model traverses a hysteresis loop between warm ice-free conditions and cold glacial conditions in response to ±5 W m−2 variations in global, annual-mean insolation. Comparison of several model configurations confirms the importance of polar ocean processes in setting the sensitivity and time scales of the transitions. A “sawtooth” character is found with faster warming and slower cooling, reflecting the opposing effects of surface heating and cooling on upper-ocean buoyancy and, thus, effective heat capacity. The transition from a glacial to warm, equable climate occurs in about 200 years. In contrast to the “freshwater hosing” scenario, transitions are driven by radiative forcing and sea ice feedbacks. The ocean circulation, and notably the meridional overturning circulation (MOC), does not drive the climate change. The MOC (and associated heat transport) collapses poleward of the advancing ice edge, but this is a purely passive response to cooling and ice expansion. The MOC does, however, play a key role in setting the time scales of the transition and contributes to the asymmetry between warming and cooling.

Hypersensitivity and growth adaptation of oestrogen-deprived MCF-7 human breast cancer cells

Background: Efficacy of endocrine therapy is compromised when human breast cancer cells circumvent imposed growth inhibition. The model of long-term oestrogen-deprived MCF-7 human breast cancer cells has suggested the mechanism results from hypersensitivity to low levels of residual oestrogen. Materials and methods: MCF-7 cells were maintained for up to 30 weeks in phenol-red-free medium and charcoal-stripped serum with 10-8 M 17-oestradiol and 10 g/ml insulin (stock 1), 10-8 M 17-oestradiol (stock 2), 10 g/ml insulin (stock 3) or no addition (stock 4). Results: Loss of growth response to oestrogen was observed only in stock 4 cells. Long-term maintenance with insulin in the absence of oestradiol (stock 3) resulted in raised oestrogen receptor alpha (ERlevels (measured by western immunoblotting) and development of hypersensitivity (assayed by oestrogen-responsive reporter gene induction and dose response to oestradiol for proliferation under serum-free conditions), but with no loss of growth response to oestrogen. Conclusion: Hypersensitivity can develop without any growth adaptation and therefore is not a prerequisite for loss of growth response in MCF-7 cells.

The global effect of follicle-stimulating hormone and tumour necrosis factor α on gene expression in cultured bovine ovarian granulosa cells

Background Oocytes mature in ovarian follicles surrounded by granulosa cells. During follicle growth, granulosa cells replicate and secrete hormones, particularly steroids close to ovulation. However, most follicles cease growing and undergo atresia or regression instead of ovulating. To investigate the effects of stimulatory (follicle-stimulating hormone; FSH) and inhibitory (tumour necrosis factor alpha; TNFα) factors on the granulosa cell transcriptome, bovine ovaries were obtained from a local abattoir and pools of granulosa cells were cultured in vitro for six days under defined serum-free conditions with treatments present on days 3–6. Initially dose–response experiments (n = 4) were performed to determine the optimal concentrations of FSH (0.33 ng/ml) and TNFα (10 ng/ml) to be used for the microarray experiments. For array experiments cells were cultured under control conditions, with FSH, with TNFα, or with FSH plus TNFα (n = 4 per group) and RNA was harvested for microarray analyses. Results Statistical analysis showed primary clustering of the arrays into two groups, control/FSH and TNFα/TNFα plus FSH. The effect of TNFα on gene expression dominated that of FSH, with substantially more genes differentially regulated, and the pathways and genes regulated by TNFα being similar to those of FSH plus TNFα treatment. TNFα treatment reduced the endocrine activity of granulosa cells with reductions in expression of FST, INHA, INBA and AMH. The top-ranked canonical pathways and GO biological terms for the TNFα treatments included antigen presentation, inflammatory response and other pathways indicative of innate immune function and fibrosis. The two most significant networks also reflect this, containing molecules which are present in the canonical pathways of hepatic fibrosis/hepatic stellate cell activation and transforming growth factor β signalling, and these were up regulated. Upstream regulator analyses also predicted TNF, interferons γ and β1 and interleukin 1β. Conclusions In vitro, the transcriptome of granulosa cells responded minimally to FSH compared with the response to TNFα. The response to TNFα indicated an active process akin to tissue remodelling as would occur upon atresia. Additionally there was reduction in endocrine function and induction of an inflammatory response to TNFα that displays features similar to immune cells.

Intercomparison of shortwave radiative transfer schemes in global aerosol modeling: results from the AeroCom Radiative Transfer Experiment

In this study we examine the performance of 31 global model radiative transfer schemes in cloud-free conditions with prescribed gaseous absorbers and no aerosols (Rayleigh atmosphere), with prescribed scattering-only aerosols, and with more absorbing aerosols. Results are compared to benchmark results from high-resolution, multi-angular line-by-line radiation models. For purely scattering aerosols, model bias relative to the line-by-line models in the top-of-the atmosphere aerosol radiative forcing ranges from roughly −10 to 20%, with over- and underestimates of radiative cooling at lower and higher solar zenith angle, respectively. Inter-model diversity (relative standard deviation) increases from ~10 to 15% as solar zenith angle decreases. Inter-model diversity in atmospheric and surface forcing decreases with increased aerosol absorption, indicating that the treatment of multiple-scattering is more variable than aerosol absorption in the models considered. Aerosol radiative forcing results from multi-stream models are generally in better agreement with the line-by-line results than the simpler two-stream schemes. Considering radiative fluxes, model performance is generally the same or slightly better than results from previous radiation scheme intercomparisons. However, the inter-model diversity in aerosol radiative forcing remains large, primarily as a result of the treatment of multiple-scattering. Results indicate that global models that estimate aerosol radiative forcing with two-stream radiation schemes may be subject to persistent biases introduced by these schemes, particularly for regional aerosol forcing.