Cobalt and Zinc Compounds Bearing 1,10-Phenanthroline-5,6-dione or 1,3,5-Triaza-7-phosphaadamantane Derivatives - Synthesis, Characterization, Cytotoxicity, and Cell Selectivity Studies

The compounds [mPTA][CoCl4] (1, mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation), [CoCl(H2O)(DION)(2)][BF4] (2, DION = 1,10-phenanthroline-5,6-dione), [Zn(DION)(2)]Cl-2 (3) and [ZnCl(O-PTA=O)(DION)][BF4] (4) were synthesized by reaction of CoCl2 with [mPTA]I or DION and ZnCl2 with DION or 1,3,5-triaza-7-phosphaadamantane-7-oxide (PTA=O) and DION, respectively. All complexes are water soluble and have been characterized by IR, far-IR, H-1, C-13 and P-31{H-1} NMR spectroscopy, ESI-MS, elemental analyses and single-crystal X-ray diffraction structural analysis (for 1). They were screened against the human tumour cell lines HCT116, HepG2 and MCF7. Complexes 2 and 3 exhibit the highest in vitro cytotoxicity and show lower cytotoxic activities in normal human fibroblast cell line than in HCT116 tumour cell line, which demonstrates their slight specificity for this type of tumour cell.

Transport mechanism in ionic liquid membranes and the study of thiomersal biodegradation

The initial goal of this work was the development of a supported liquid membrane (SLM) bioreactor for the remediation of vaccine production effluents contaminated with a highly toxic organomercurial – thiomersal. Therefore, two main aspects were focused on: 1) the development of a stable supported liquid membrane – using room temperature ionic liquids (RTILs) – for the selective transport of thiomersal from the wastewater to a biological compartment, 2) study of the biodegradation kinetics of thiomersal to metallic mercury by a Pseudomonas putida strain. The first part of the work focused on the evaluation of the physicochemical properties of ionic liquids and on the SLMs’ operational stability. The results obtained showed that, although it is possible to obtain a SLM with a high stability, water possesses nonnegligible solubility in the RTILs studied. The formation of water clusters inside the hydrophobic ionic liquid was identified and found to regulate the transport of water and small ions. In practical terms, this meant that, although it was possible to transport thiomersal from the vaccine effluent to the biological compartment, complete isolation of the microbial culture could not be guaranteed and the membrane might ultimately be permeable to other species present in the aqueous vaccine wastewater. It was therefore decided not to operate the initially targeted integrated system but, instead, the biological system by itself. Additionally, attention was given to the development of a thorough understanding of the transport mechanisms involved in the solubilisation and transport of water through supported liquid membranes with RTILs as well as to the evaluation of the effect of water uptake by the SLM in the transport mechanisms of water-soluble solutes and its effect on SLM performance. The results obtained highlighted the determinant role played by water – solubilised inside the ionic liquids – on the transport mechanism. It became clear that the transport mechanism of water and water-soluble solutes through SLMs with [CnMIM][PF6] RTILs was regulated by the dynamics of water clusters inside the RTIL, rather than by molecular diffusion through the bulk of the ionic liquid. Although the stability tests vi performed showed that there were no significant losses of organic phase from the membrane pores, the formation of water clusters inside the ionic liquid, which constitute new, non-selective environments for solute transport, leads to a clear deterioration of SLM performance and selectivity. Nevertheless, electrical impedance spectroscopy characterisation of the SLMs showed that the formation of water clusters did not seem to have a detrimental effect on the SLMs’ electrical characteristics and highlighted the potential of using this type of membranes in electrochemical applications with low resistance requirements. The second part of the work studied the kinetics of thiomersal degradation by a pure culture of P. putida spi3 strain, in batch culture and using a synthe tic wastewater. A continuous ly stirred tank reactor fed with the synthetic wastewater was also operated and the bioreactor’s performance and robustness, when exposed to thiomersal shock loads, were evaluated. Finally, a bioreactor for the biological treatment of a real va ccine production effluent was set up and operated at different dilution rates. Thus it was possible to treat a real thiomersal-contaminated effluent, lowering the outlet mercury concentration to values below the European limit for mercury effluent discharges.

Assessment of nutritional and metabolic profiles of pea shoots: The new ready-to-eat baby-leaf vegetable

Pea-shoots are a new option as ready-to-eat baby-leaf vegetable. However, data about the nutritional composition and the shelf-life stability of these leaves, especially their phytonutrient composition is scarce. In this work, the macronutrient, micronutrient and phytonutrients profile of minimally processed pea shoots were evaluated at the beginning and at the end of a 10-day storage period. Several physicochemical characteristics (color, pH, total soluble solids, and total titratable acidity) were also monitored. Standard AOAC methods were applied in the nutritional value evaluation, while chromatographic methods with UV–vis and mass detection were used to analyze free forms of vitamins (HPLC-DAD-ESI-MS/MS), carotenoids (HPLC-DAD-APCI-MSn) and flavonoid compounds (HPLC-DAD-ESI-MSn). Atomic absorption spectrometry (HR-CS-AAS) was employed to characterize the mineral content of the leaves. As expected, pea leaves had a high water (91.5%) and low fat (0.3%) and carbohydrate (1.9%) contents, being a good source of dietary fiber (2.1%). Pea shoots showed a high content of vitamins C, E and A, potassium and phosphorous compared to other ready-to-eat green leafy vegetables. The carotenoid profile revealed a high content of β-carotene and lutein, typical from green leafy vegetables. The leaves had a mean flavonoid content of 329 mg/100 g of fresh product, mainly composed by glycosylated quercetin and kaempferol derivatives. Pea shoots kept their fresh appearance during the storage being color maintained throughout the shelf-life. The nutritional composition was in general stable during storage, showing some significant (p < 0.05) variation in certain water-soluble vitamins.

Fresh-cut aromatic herbs: Nutritional quality stability during shelf-life

Fresh-cut vegetables are a successful convenient healthy food. Nowadays, the presence of new varieties of minimally processed vegetables in the market is common in response to the consumers demand for new flavours and high quality products. Within the most recent fresh-cut products are the aromatic herbs. In this work, the objective was to evaluate the nutritional quality and stability of four fresh-cut aromatic herbs. Several physicochemical quality characteristics (colour, pH, total soluble solids, and total titratable acidity) were monitored in fresh-cut chives, coriander, spearmint and parsley leaves, stored under refrigeration (3 ± 1 ºC) during 10 days. Their nutritional composition was determined, including mineral composition (phosphorous, potassium, sodium, calcium, magnesium, iron, zinc, manganese and copper) and fat- and water-soluble vitamin contents. Total soluble phenolics, flavonoids and the antioxidant capacity were determined by spectrophotometric methods. The aromatic herbs kept their fresh appearance during the storage, maintaining their colour throughout shelf-life. Their macronutrient composition and mineral content were stable during storage. Coriander had the highest mineral and fatsoluble vitamin content, while spearmint showed the best scores in the phenolic, flavonoid and antioxidant capacity assays. Vitamins and antioxidant capacity showed some variation during storage, with a differential behaviour of each compound according to the sample.

Valorization of grape pomace through hot compressed water extraction/hydrolysis

The world energy consumption is expected to increase strongly in coming years, because of the emerging economies. Biomass is the only renewable carbon resource that is abundant enough to be used as a source of energy Grape pomace is one of the most abundant agro-industrial residues in the world, being a good biomass resource. The aim of this work is the valorization of grape pomace from white grapes (WWGP) and from red grapes (RWGP), through the extraction of phenolic compounds with antioxidant activity, as well as through the extraction/hydrolysis of carbohydrates, using subcritical water, or hot compressed water (HCW). The main focus of this work is the optimization of the process for WWGP, while for RWGP only one set of parameters were tested. The temperatures used were 170, 190 and 210 °C for WWGP, and 180 °C for RWGP. The water flow rates were 5 and 10 mL/min, and the pressure was always kept at 100 bar. Before performing HCW assays, both residues were characterized, revealing that WWGP is very rich in free sugars (around 40%) essentially glucose and fructose, while RWGP has higher contents of structural sugars, lignin, lipids and protein. For WWGP the best results were achieved at 210 °C and 10 mL/min: higher yield in water soluble compounds (69 wt.%), phenolics extraction (26.2 mg/g) and carbohydrates recovery (49.3 wt.% relative to the existing 57.8%). For RWGP the conditions were not optimized (180 °C and 5 mL/min), and the values of the yield in water soluble compounds (25 wt.%), phenolics extraction (19.5 mg/g) and carbohydrates recovery (11.4 wt.% relative to the existing 33.5%) were much lower. The antioxidant activity of the HCW extracts from each assay was determined, the best result being obtained for WWGP, namely for extracts obtained at 210 °C (EC50=20.8 μg/mL; EC50 = half maximum effective concentration; EC50 = 22.1 μg/mL for RWGP, at 180 ºC).

Effect of surface plasmon resonance in TiO2/Au thin films on the fluorescence of self-assembled CdTe QDs structure

The exceptional properties of localised surface plasmons (LSPs), such as local field enhancement and confinement effects, resonant behavior, make them ideal candidates to control the emission of luminescent nanoparticles. In the present work, we investigated the LSP effect on the steady-state and time-resolved emission properties of quantum dots (QDs) by organizing the dots into self-assembled dendrite structures deposited on plasmonic nanostructures. Self-assembled structures consisting of water-soluble CdTe mono-size QDs, were developed on the surface of co-sputtered TiO2 thin films doped with Au nanoparticles (NPs) annealed at different temperatures. Their steady-state fluorescence properties were probed by scanning the spatially resolved emission spectra and the energy transfer processes were investigated by the fluorescence lifetime imaging (FLIM) microscopy. Our results indicate that a resonant coupling between excitons confined in QDs and LSPs in Au NPs located beneath the self-assembled structure indeed takes place and results in (i) a shift of the ground state luminescence towards higher energies and onset of emission from excited states in QDs, and (ii) a decrease of the ground state exciton lifetime (fluorescence quenching).

Predator-prey chemical warfare determines the expression of biocontrol genes by rhizosphere-associated Pseudomonas fluorescens.

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.

Basic biochemical investigations as rationale for the design of original antimalarial drugs. An example of phospholipid metabolism

The future of antimalarial chemotherapy is particulary alarming in view of the spread of parasite cross-resistances to drugs that are not even structurally related. Only the availability of new pharmacological models will make it possible to select molecules with novel mechanisms of action, thus delaving resistance and allowing the development of new chemotherapeutic strategies. We reached this objective in mice. Our approach is hunged on fundamental and applied research begun in 1980 to investigate to phospholipid (PL) metabolism of intraerythrocytic Plasmodium. This metabolism is abundant, specific and indispensable for the production of Plasmodium membranes. Any drug to interfere with this metabolism blocks parasitic development. The most effective interference yet found involves blockage of the choline transporter, which supplies Plasmodium with choline for the synthesis of phosphatidylcholine, its major PL, this is a limiting step in the pathway. The drug sensitivity thereshold is much lower for the parasite, which is more dependent on this metabolism than host cells. The compounds show in vitro activity against P. falciparum at 1 to 10 nM. They show a very low toxicity against a lymphblastoid cell line, demonstrating a total abscence of correlation between growth inhibition of parasites and lymphoblastoid cells. They show antimalarial activity in vivo, in the P. berghei or P. chabaudi/mouse system, at doses 20-to 100-fold lower than their in acute toxicity limit. The bioavailability of a radiolabeled form of the product seemed to be advantageous (slow blood clearance and no significant concentration in tissues). Lastly, the compounds are inexpensive to produce. They are stable and water-soluble.

Antibody isotypes, including IgG subclasses, in Ecuadorian patients with pulmonary Paragonimiasis

An ELISA test was developed to detect Paragonimus-specific antibodies, including IgG subclasses, using P. mexicanus crude water-soluble antigens. The test was standardized to detect antibodies in sera of Ecuadorian patients with pulmonary paragonimiasis and negative controls from the endemic area. The detected mean levels of IgG (0.753, SEM: 0.074) and IgM (0.303, SEM: 0.033) were significantly elevated (P<0.05). Within the IgG subclasses, IgG4 showed the highest detected mean level (0.365, SEM: 0.116) and the other three subclasses showed considerably lower mean levels (IgG1, 0.186 SEM: 0.06; IgG2, 0.046 SEM: 0.01; IgG3, 0.123 SEM: 0.047). The number of P. mexicanus eggs found in sputum of infected individuals showed a positive correlation with the level of antibodies detected for IgM, IgG and its subclasses (P<0.001). The relevance of these findings in Ecuadorian patients suffering from pulmonary paragonimiasis is discussed.

Organometallic cages as vehicles for intracellular release of photosensitizers.

Water-soluble metalla-cages were used to deliver hydrophobic porphin molecules to cancer cells. After internalization, the photosensitizer was photoactivated, significantly increasing the cytotoxicity in cells. During the transport, the photosensitizer remains nonreactive to light, offering a new strategy to tackle overall photosensitization, a limitation often encountered in photodynamic therapy.

The glycoinositol-phospholipids of Phytomonas.

The Phytomonas spp. are trypanosomatid parasites of plants. A polar glycolipid fraction of a Phytomonas sp., isolated from the plant Euphorbia characias and grown in culture, was fractionated into four major glycolipid species (Phy 1-4). The glycolipids were analysed by chemical and enzymic modifications, composition and methylation analyses, electrospray mass spectrometry and microsequencing after HNO2 deamination and NaB3H4 reduction. The water-soluble headgroup of the Phy2 glycolipid was also analysed by 1H NMR. All four glycolipids were shown to be glycoinositol-phospholipids (GIPLs) with phosphatidylinositol (PI) moieties containing the fully saturated alkylacylglycerol lipids 1-O-hexadecyl-2-O-palmitoylglycerol and 1-O-hexadecyl-2-O-stearoylglycerol. The structures of the Phy 1-4 GIPLs are: Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI, Glc alpha 1-2(NH2-CH2CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6PI, [formula: see text] Glc alpha 1-2(NH2CH2CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4(NH2-CH2CH2-HPO4-)GlcN alpha 1-6PI [formula: see text] and Glc alpha 1-2Glc alpha 1-2(NH2CH2-CH2-HPO4-)Man alpha 1-2Man alpha 1-6Man alpha 1-4(NH2CH2CH2-HPO4-)-GlcN alpha 1-6PI. [formula: see text] The Phytomonas GIPLs represent a novel series of structures. This is the first description of the chemical structure of cell-surface molecules of this plant pathogen. The Phytomonas GIPLs are compared with those of other trypanosomatid parasites and are discussed with respect to trypanosomatid phylogenetic relationships.

Vitaminas B y homocisteína en la insuficiencia renal crónica

Metabolic, biochemical, and hormonal changes occur in chronic renal failure usually associated with hyponutrition states. In predialysis patients, knowing the nutritional state about water-soluble vitamins such as thiamine, riboflavin, pyridoxine, cianocobalamine, and folic acid is becoming more and more important since some of the manifestations of chronic renal failure may be due to the deficiency of some of these water-soluble vitamins. The metabolic pathways in which most of these vitamins participate are interrelated and it is difficult to understand how the individual deficits of each vitamin affect renal pathology. This work aims at reviewing not only this issue but also the status of these water-soluble vitamins that different authors have found in groups of predialysis patients. On the other hand, the issue on the high prevalence of hyperhomocysteinemia in chronic renal failure as the main mortality risk factor due to cardiovascular pathologies as well as the implication of these vitamins in the metabolism of homocysteine, and consequently in plasma levels of this metabolite in predialysis patients is reviewed.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

Soil phosphorus fractions in sandy soils amended with cattle manure for long periods

Phosphorus fractions were determined in soil samples from areas fertilized or not with farmyard cattle manure (FYM) and in samples of FYM used in the semi-arid region of Paraiba state, Brazil. Soil samples were taken from the 0-20; 20-40 and 40-60 cm layers of 18 cultivated areas, which, according to interviews with farmers, had been treated with 12 to 20 t ha-1 FYM annually, for the past 2 to 40 years. Soil samples were also collected from four unfertilized pasture areas as controls. Phosphorus in the soil samples was sequentially extracted with water (Pw), resin (Pres), NaHCO3 (Pi bic and Po bic), NaOH (Pi hid and Po hid), H2SO4 (Pacid) and, finally, by digestion with H2SO4/H2O2 (Presd). Nine FYM samples were extracted with water, resin, Mehlich-1, H2SO4, NaOH or digestion with H2SO4/H2O2, not sequentially, and the extracts analyzed for P. The sampled areas had homogeneous, sandy and P-deficient soils; increases in total soil P (Pt) above the mean value of the control areas (up to 274 mg kg-1 in the 0-20 cm layer of the most P-enriched samples) were therefore attributed to FYM applications, which was the only external P input in the region. Regression analysis was used to study the relationship between soil P fractions and Pt. The Pacid fraction, related to Ca-P forms, showed the greatest increases (p < 0.01) as a result of FYM applications, rising from 8.4 mg kg-1 in a non-fertilized sample to 43.8 mg kg-1 in the sample with the highest Pt content. The sum of Pw, Pres and Pi bic, considered as labile P, showed comparable increases with Pacid, while Pi hid showed the smallest increase due to FYM applications. Organic P forms also increased, more so the fraction Po hid, considered less labile, than the more labile one, Po bic. The residual P fraction was practically half of Pt, independently of the Pt value. Increases in labile P, Pacid and organic P were justified by the high average concentration of Pw (36 %), Pacid (34 %), and Po hid (30 %) in the FYM. Significant changes in the proportion of P forms among soil layers indicated the downward movement of P in organic forms.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.