Evaluation of Cellular Uptake and Removal of Chlorpropham in the Treatment of Dunaliella salina for Phytoene Production.

Chlorpropham is a carbamate herbicide that inhibits cell division and has been widely used as a potato sprout suppressant. Recently we showed that the microalga Dunaliella salina treated with chlorpropham massively accumulated the colourless carotenoids phytoene and phytofluene. Phytoene and phytofluene are valued for their antioxidant, UV-absorption and skin protectant properties; however, they are present in very low quantities in nature. The low toxicity herbicide chlorpropham seems a promising catalyst to produce phytoene in large quantities from CO2 and solar energy with D. salina. This study explored chlorpropham uptake by the algal cells, the formation of potential intermediate metabolites, and the removal of residual chlorpropham from harvested D. salina biomass. Algal biomass rapidly concentrated chlorpropham from culture media. However, washing the harvested biomass with fresh culture medium twice and five times removed ~83 and ~97% of the chlorpropham from the biomass, respectively, and retained algal cell integrity. Furthermore, chloroaniline, a common metabolite of chlorpropham degradation, was not detected in chlorpropham-treated cultures, which were monitored every two days for thirty days. Cells treated with chlorpropham for either 10 min or 24 h continued to over-accumulate phytoene after resuspension in an herbicide-free medium. These data imply that whilst Dunaliella cells do not possess the intracellular capacity to degrade chlorpropham to chloroaniline, the effect of chlorpropham is irreversible on cell nuclear division and hence on carotenoid metabolism.

A Comparison of ß-Carotene, Phytoene and Amino Acids Production in Dunaliella salina DF 15 (CCAP 19/41) and Dunaliella salina CCAP 19/30 Using Different Light Wavelengths.

Strains of Dunaliella salina microalgae are of considerable research and industrial interest because they hyper-accumulate ß-carotene as well as produce high-quality protein. To explore the co-production of valuable compounds in D. salina, this study compared the production of ß-carotene, phytoene and amino acids in two strains cultivated under white, red or blue light until no further nitrogen was available. D. salina DF15 (CCAP 19/41 (PLY DF15)) produced more than 12% ß-carotene (ash-free dry weight (AFDW) basis), and red light triggered the production of 9-cis ß-carotene at a 9-cis/all-trans ß-carotene ratio of 1.5. Phytoene production was also evident in D. salina DF15 under all conditions, particularly under blue light. However, the profile of essential amino acids (EAAs) and calculation of the essential amino acid index (EAAI) was less than ideal in terms of protein quality, for both strains. Umami compounds, quantified as monosodium glutamate (MSG) equivalents, indicated a higher equivalent umami concentration (EUC) in D. salina DF15 under red light (3.2 g MSG/100 g AFDW) than in D. salina CCAP19/30. Overall, D. salina DF15 demonstrates valuable traits for further exploration and product optimisation.

Mitosis Inhibitors Induce Massive Accumulation of Phytoene in the Microalga Dunaliella salina.

Phytoene is a colourless natural carotenoid that absorbs UV light and provides antioxidant and anti-inflammatory activities as well as protection against photodamage. It is therefore valued for its skin health and aesthetic benefits by the cosmetic industry, as well as by the health food sector. The halotolerant green microalga Dunaliella salina is one of the richest sources of natural carotenoids. We have previously investigated the over-production of phytoene in D. salina after cultivation with the well-characterised mitosis inhibitor, chlorpropham. In this study, 15 herbicides with different modes of action were tested for their potential to promote phytoene accumulation. All herbicides showed different levels of capabilities to support phytoene over-production in D. salina. Most significantly, the two mitosis inhibitors tested in this study, propyzamide and chlorpropham, showed similar capacities to support the over-production of phytoene by D. salina cultures as phytoene desaturase inhibitors. The cellular content of phytoene increased by over 10-fold within 48 h of treatment with the mitosis inhibitors compared to untreated cultures. Results indicate a general effect of mitosis inhibitors on phytoene accumulation in D. salina. Furthermore, red light was found to significantly enhance the phytoene yield when used in combination with effective inhibitor treatments. Red light can be applied to maximize the production of phytoene from D. salina.

Effect of Light Intensity and Wavelength on Biomass Growth and Protein and Amino Acid Composition of Dunaliella salina.

Dunaliella salina is a halotolerant, photoautotrophic marine microalga and one of the richest sources of natural carotenoids but also shows potential as a novel food source with high protein quality. This study sought to optimise the production of biomass, protein and amino acids from D. salina, alongside carotenoids using a two-stage cultivation approach based on the use of light of different intensities and quality, i.e., white, red and blue LED light. In stage 1, four white LED light intensities were tested. In stage 2, the same four light intensities from either blue or red LEDs were applied once exponential growth ceased and cells reached the stationary phase under white LED light in stage 1. Remarkably, both biomass concentration and biomass productivity showed a 1.3-1.7-fold increase in stage 2, without medium replenishment, while protein concentration and protein productivity showed an ~1.1-fold increase. The amino acid content and amino acid index remained unchanged from stage 1 to stage 2, and minimum difference was found across different light intensities. Overall, D. salina delivered so-called high protein quality, with an essential amino acid index (EAAI) of 0.99, and red light, which has previously been shown to increase carotenoid production, boosted further biomass production over and above white light, at all light intensities tested.

Carotenoid Production by Dunaliella salina under Red Light.

The halotolerant photoautotrophic marine microalga Dunaliella salina is one of the richest sources of natural carotenoids. Here we investigated the effects of high intensity blue, red and white light from light emitting diodes (LED) on the production of carotenoids by strains of D. salina under nutrient sufficiency and strict temperature control favouring growth. Growth in high intensity red light was associated with carotenoid accumulation and a high rate of oxygen uptake. On transfer to blue light, a massive drop in carotenoid content was recorded along with very high rates of photo-oxidation. In high intensity blue light, growth was maintained at the same rate as in red or white light, but without carotenoid accumulation; transfer to red light stimulated a small increase in carotenoid content. The data support chlorophyll absorption of red light photons to reduce plastoquinone in photosystem II, coupled to phytoene desaturation by plastoquinol:oxygen oxidoreductase, with oxygen as electron acceptor. Partitioning of electrons between photosynthesis and carotenoid biosynthesis would depend on both red photon flux intensity and phytoene synthase upregulation by the red light photoreceptor, phytochrome. Red light control of carotenoid biosynthesis and accumulation reduces the rate of formation of reactive oxygen species (ROS) as well as increases the pool size of anti-oxidant.

Mitochondrial Function, Mobility and Lifespan Are Improved in Drosophila melanogaster by Extracts of 9-cis-ß-Carotene from Dunaliella salina.

Carotenoids are implicated in alleviating ageing and age-related diseases in humans. While data from different carotenoids are mixed in their outcomes, those for 9-cis-ß-carotene indicate general positive effects, although basic data on its biological impact are limited. Here, we show that supplementation with 9-cis-ß-carotene in ageing Drosophila melanogaster improved mitochondrial function in terms of ATP production and whole-body respiration and extended mean lifespan. It also resulted in improved mobility. These data provide a potential biological rational for the beneficial effects of dietary supplementation with 9-cis-ß-carotene. These effects may be based on the maintenance of a sound mitochondrial function.

Red Light Control of ß-Carotene Isomerisation to 9-cis ß-Carotene and Carotenoid Accumulation in Dunaliella salina.

Dunaliella salina is a rich source of 9-cis ß-carotene, which has been identified as an important biomolecule in the treatment of retinal dystrophies and other diseases. We previously showed that chlorophyll absorption of red light photons in D. salina is coupled with oxygen reduction and phytoene desaturation, and that it increases the pool size of ß-carotene. Here, we show for the first time that growth under red light also controls the conversion of extant all-trans ß-carotene to 9-cis ß-carotene by ß-carotene isomerases. Cells illuminated with red light from a light emitting diode (LED) during cultivation contained a higher 9-cis ß-carotene content compared to cells illuminated with white or blue LED light. The 9-cis/all-trans ß-carotene ratio in red light treated cultures reached >2.5 within 48 h, and was independent of light intensity. Illumination using red light filters that eliminated blue wavelength light also increased the 9-cis/all-trans ß-carotene ratio. With norflurazon, a phytoene desaturase inhibitor which blocked downstream biosynthesis of ß-carotene, extant all-trans ß-carotene was converted to 9-cis ß-carotene during growth with red light and the 9-cis/all-trans ß-carotene ratio was ~2. With blue light under the same conditions, 9-cis ß-carotene was likely destroyed at a greater rate than all-trans ß-carotene (9-cis/all-trans ratio 0.5). Red light perception by the red light photoreceptor, phytochrome, may increase the pool size of anti-oxidant, specifically 9-cis ß-carotene, both by upregulating phytoene synthase to increase the rate of biosynthesis of ß-carotene and to reduce the rate of formation of reactive oxygen species (ROS), and by upregulating ß-carotene isomerases to convert extant all-trans ß-carotene to 9-cis ß-carotene.

Potential of New Isolates of Dunaliella Salina for Natural ß-Carotene Production.

The halotolerant microalga Dunaliella salina has been widely studied for natural ß-carotene production. This work shows biochemical characterization of three newly isolated Dunaliellasalina strains, DF15, DF17, and DF40, compared with D. salina CCAP 19/30 and D. salina UTEX 2538 (also known as D. bardawil). Although all three new strains have been genetically characterized as Dunaliella salina strains, their ability to accumulate carotenoids and their capacity for photoprotection against high light stress are different. DF15 and UTEX 2538 reveal great potential for producing a large amount of ß-carotene and maintained a high rate of photosynthesis under light of high intensity; however, DF17, DF40, and CCAP 19/30 showed increasing photoinhibition with increasing light intensity, and reduced contents of carotenoids, in particular ß-carotene, suggesting that the capacity of photoprotection is dependent on the cellular content of carotenoids, in particular ß-carotene. Strong positive correlations were found between the cellular content of all-trans ß-carotene, 9-cis ß-carotene, all-trans α-carotene and zeaxanthin but not lutein in the D. salina strains. Lutein was strongly correlated with respiration in photosynthetic cells and strongly related to photosynthesis, chlorophyll and respiration, suggesting an important and not hitherto identified role for lutein in coordinated control of the cellular functions of photosynthesis and respiration in response to changes in light conditions, which is broadly conserved in Dunaliella strains. Statistical analysis based on biochemical data revealed a different grouping strategy from the genetic classification of the strains. The significance of these data for strain selection for commercial carotenoid production is discussed.

Potential process 'hurdles' in the use of macroalgae as feedstock for biofuel production in the British Isles.

This review examines the potential technical and energy balance hurdles in the production of seaweed biofuel, and in particular for the MacroBioCrude processing pipeline for the sustainable manufacture of liquid hydrocarbon fuels from seaweed in the UK. The production of biofuel from seaweed is economically, energetically and technically challenging at scale. Any successful process appears to require both a method of preserving the seaweed for continuous feedstock availability and a method exploiting the entire biomass. Ensiling and gasification offer a potential solution to these two requirements. However there is need for more data particularly at a commercial scale. © 2016 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Tyrosine Kinase Inhibitors. 20. Optimization of Substituted Quinazoline and Pyrido[3,4-d]pyrimidine Derivatives as Orally Active, Irreversible Inhibitors of the Epidermal Growth Factor Receptor Family.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of quinazoline- and pyrido[3,4-d]pyrimidine-based analogues of the irreversible pan-erbB inhibitor, canertinib. Cyclic amine bearing crotonamides were determined to provide rapid inhibition of cellular erbB1 autophosphorylation and good metabolic stability in liver microsome and hepatocyte assays. The influence of 4-anilino substitution on pan-erbB inhibitory potency was investigated. Several anilines were identified as providing potent, reversible pan-erbB inhibition. Optimum 4- and 6-substituents with known 7-substituents provided preferred irreversible inhibitors for pharmacodynamic testing in vivo. Quinazoline 54 and pyrido[3,4-d]pyrimidine 71 were identified as clearly superior to canertinib. Both compounds possess a piperidinyl crotonamide Michael acceptor and a 3-chloro-4-fluoroaniline, indicating these as optimized 6- and 4-substituents, respectively. Pharmacokinetic comparison of compounds 54 and 71 across three species selected compound 54 as the preferred candidate. Compound 54 (PF-00299804) has been assigned the nomenclature of dacomitinib and is currently under clinical evaluation.

The influence of photoperiod and light intensity on the growth and photosynthesis of Dunaliella salina (chlorophyta) CCAP 19/30.

The green microalga Dunaliella salina survives in a wide range of salinities via mechanisms involving glycerol synthesis and degradation and is exploited for large amounts of nutraceutical carotenoids produced under stressed conditions. In this study, D. salina CCAP 19/30 was cultured in varying photoperiods and light intensities to study the relationship of light with different growth measurement parameters, with cellular contents of glycerol, starch and carotenoids, and with photosynthesis and respiration. Results show CCAP 19/30 regulated cell volume when growing under light/dark cycles: cell volume increased in the light and decreased in the dark, and these changes corresponded to changes in cellular glycerol content. The decrease in cell volume in the dark was independent of cell division and biological clock and was regulated by the photoperiod of the light/dark cycle. When the light intensity was increased to above 1000 µmol photons m(-2) s(-1), cells displayed evidence of photodamage. However, these cells also maintained the maximum level of photosynthesis efficiency and respiration possible, and the growth rate increased as light intensity increased. Significantly, the intracellular glycerol content also increased, >2-fold compared to the content in light intensity of 500 µmol photons m(-2) s(-1), but there was no commensurate increase in the pool size of carotenoids. These data suggest that in CCAP 19/30 glycerol stabilized the photosynthetic apparatus for maximum performance in high light intensities, a role normally attributed to carotenoids.

Emerging pollutants and plants--Metabolic activation of diclofenac by peroxidases.

Human pharmaceuticals and their residues are constantly detected in our waterbodies, due to poor elimination rates, even in the most advanced waste water treatment plants. Their impact on the environment and human health still remains unclear. When phytoremediation is applied to aid water treatment, plants may transform and degrade xenobiotic contaminants through phase I and phase II metabolism to more water soluble and less toxic intermediates. In this context, peroxidases play a major role in activating compounds during phase I via oxidation. In the present work, the ability of a plant peroxidase to oxidize the human painkiller diclofenac was confirmed using stopped flow spectroscopy in combination with LC-MS analysis. Analysis of an orange colored product revealed the structure of the highly reactive Diclofenac-2,5-Iminoquinone, which may be the precursor of several biological conjugates and breakdown products in planta.

Responses of tall fescue (Festuca arundinacea) to growth in naphthalene-contaminated sand: xenobiotic stress versus water stress.

The adaptations of tall fescue (Festuca arundinacea) arising from growth in naphthalene-contaminated sand (0.8 g kg(-1) sand dry weight (dw)) were investigated in the contexts of xenobiotic stress and water stress. The transfer of polycyclic aromatic hydrocarbons (PAHs) across the root endodermis was investigated using the hydrophobic Nile red stain as a PAH homologue. Nile red was applied to the epidermis of a living root to visualise uptake into the root through the transpiration stream, and the distance travelled by the stain into the root tissues was investigated using epi-fluorescence microscopy (Nikon Eclipse 90i). The results showed that the Nile red applied to the roots grown in naphthalene-contaminated sand was unable to penetrate the roots beyond the endodermis, whereas those grown in 'clean' sand showed evidence of uptake into the xylem vessels beyond the endodermis. Furthermore, partial collapse was observed in the cortex of naphthalene-treated roots, suggesting drought stress. Interestingly, the treated plants showed visual resilience to drought stress whilst the leaves of the control plants showed signs of wilting.

Changes in the abundance of sugars and sugar-like compounds in tall fescue (Festuca arundinacea) due to growth in naphthalene-treated sand.

The hydrophilic metabolome of tall fescue (Festuca arundinacea) adapted to grow in naphthalene-treated sand (0.8 g kg(-1) sand dw) was analysed using gas chromatography-mass spectrometry, and peaks corresponding to the more abundant compounds were tentatively identified from analysis of their mass spectral features and reference to the NIST Mass Spectral Database. Particular attention was paid to sugars as they are known to play important roles as stress regulators in plants. The results showed that the abundance of sugars was greater in the roots but lesser in the shoots of treated plants when compared to their control counterparts. The results for indole acetic acid (IAA) were notable: IAA was prominently less in the treated roots compared to shoots, and in treated shoots, IAA was particularly subdued compared to untreated shoots consistent with IAA degradation in treated plant tissues. The differences in the molecular phenotype between control and treated plants were expressed in root structural differences. The treated roots were modified to have greater suberisation, enhanced thickening in the endodermis and distortions in the cortical zone as demonstrated through scanning electron and epi-fluorescence microscopy.

Scanning electron microscopic investigations of root structural modifications arising from growth in crude oil-contaminated sand.

The choice of plant for phytoremediation success requires knowledge of how plants respond to contaminant exposure, especially their roots which are instrumental in supporting rhizosphere activity. In this study, we investigated the responses of plants with different architectures represented by beetroot (Beta vulgaris), a eudicot with a central taproot and many narrower lateral roots, and tall fescue (Festuca arundinacea), a monocot possessing a mass of threadlike fibrous roots to grow in crude oil-treated sand. In this paper, scanning electron microscopy was used to investigate modifications to plant root structure caused by growth in crude oil-contaminated sand. Root structural disorders were evident and included enhanced thickening in the endodermis, increased width of the root cortical zone and smaller diameter of xylem vessels. Inhibition in the rate of root elongation correlated with the increase in cell wall thickening and was dramatically pronounced in beetroot compared to the roots of treated fescue. The latter possessed significantly fewer (p < 0.001) and significantly shorter (p < 0.001) root hairs compared to control plants. Possibly, root hairs that absorb the hydrophobic contaminants may prevent contaminant absorption into the main root and concomitant axile root thickening by being sloughed off from roots. Tall fescue exhibited greater root morphological adaptability to growth in crude oil-treated sand than beetroot and, thus, a potential for long-term phytoremediation.

Antitumor activity and pharmacokinetic properties of PF-00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor.

Signaling through the erbB receptor family of tyrosine kinases contributes to the proliferation, differentiation, migration, and survival of a variety of cell types. Abnormalities in members of this receptor family have been shown to play a role in oncogenesis, thus making them attractive targets for anticancer treatments. PF-00299804 is a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor currently in phase I clinical trials. PF-00299804 is believed to irreversibly inhibit erbB tyrosine kinase activity through binding at the ATP site and covalent modification of nucleophilic cysteine residues in the catalytic domains of erbB family members. Oral administration of PF-00299804 causes significant antitumor activity, including marked tumor regressions in a variety of human tumor xenograft models that express and/or overexpress erbB family members or contain the double mutation (L858R/T790M) in erbB1 (EGFR) associated with resistance to gefitinib and erlotinib. Furthermore, PF-00299804 shows exceptional distribution to human tumor xenografts and excellent pharmacokinetic properties across species.

Tyrosine kinase inhibitors. 19. 6-Alkynamides of 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as irreversible inhibitors of the erbB family of tyrosine kinase receptors.

Structure-activity relationships for inhibition of erbB1, erbB2, and erbB4 were determined for a series of alkynamide analogues of quinazoline- and pyrido[3,4-d]pyrimidine-based compounds. The compounds were prepared by coupling the appropriate 6-aminoquinazolines or 6-aminopyrido[3,4-d]pyrimidines with alkynoic acids, using EDCI.HCl in pyridine. The compounds showed pan-erbB enzyme inhibition but were on average about 10-fold more potent against erbB1 than against erbB2 and erbB4. For cellular inhibition, the nature of the alkylating side chains was an important determinant, with 5-dialkylamino-2-pentynamide type Michael acceptors providing the highest potency. This is suggested to be due to an improved ability of the amine to participate in an autocatalysis of the Michael reaction with enzyme cysteine residues. Pyrido[3,4-d]pyrimidine analogue 39 was selected for in vivo evaluation and achieved tumor regressions at 10 mg/kg in the A431 human epidermoid carcinoma and at 40 mg/kg for the SF767 human glioblastoma and the SKOV3 human ovarian carcinoma. Complete stasis was observed at 40 mg/kg in the BXPC3 human pancreatic carcinoma as well as in the H125 human non-small-cell lung carcinoma.

2-Aminoquinazoline inhibitors of cyclin-dependent kinases.

The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.

Pyrido[2,3-d]pyrimidin-7-ones as specific inhibitors of cyclin-dependent kinase 4.

Inhibition of the cell cycle kinase, cyclin-dependent kinase-4 (Cdk4), is expected to provide an effective method for the treatment of proliferative diseases such as cancer. The pyrido[2,3-d]pyrimidin-7-one template has been identified previously as a privileged structure for the inhibition of ATP-dependent kinases, and good potency against Cdks has been reported for representative examples. Obtaining selectivity for individual Cdk enzymes, particularly Cdk4, has been challenging. Here, we report that the introduction of a methyl substituent at the C-5 position of the pyrido[2,3-d]pyrimidin-7-one template is sufficient to confer excellent selectivity for Cdk4 vs other Cdks and representative tyrosine kinases. Further optimization led to the identification of highly potent and selective inhibitors of Cdk4 that exhibit potent antiproliferative activity against human tumor cells in vitro. The most selective Cdk4 inhibitors were evaluated for antitumor activity against MDA-MB-435 human breast carcinoma xenografts in mice.