Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the ‘cold chain’, the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4 oC, but not when stored at 40 oC / 75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40 oC / 75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation as compared to the original formulation when stored at 40 oC /75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

ABSORPTION AND EMISSION-SPECTRA OF THE YBCO LASER PLUME

The plasma produced during laser ablation deposition of thin film YBCO has been studied by optical emission spectroscopy. There is evidence of increased YO band emission in the range 590-625 nm as the ambient oxygen gas pressure confining the plume is increased in the range 30-200 m Torr. Temporal profiles show that close to the target the plume is insensitive to ambient oxygen pressure. It is deduced that the optical emission here is excited by electron impact excitation. Further away from the target there is evidence that two distinct processes are at work. One is again electron excitation; the emission from this process decreases with distance because the expanding plume cools and collisions become less frequent in the expanding gas. The second is driven by oxidation of atomic species expelled at high speeds from the target. The main region of this activity is in the plume sheath where a shock front ensures heating of ambient O2 and reaction of monatomic plasma species to form oxide in an exothermic reaction. Spatial mapping of the emission demonstrates clearly how increasing oxygen gas pressure confines the plasma and enhances the emission intensity from the molecular YO species ejected from the target in a smaller region close to the target. Ba+ is observed as a dominant species only very close to (within 1 mm of) the target. Absorption spectra have been taken in an attempt to examine ground state and cool species in the plume. They reveal the quite surprising result that YO persists in the chamber for periods up to 1 msec. This suggests an explanation for the recent report of off-axis laser deposition in terms of simple condensation. Previously, quasi-ballistic transfer of material from target to substrate has been considered the only significant process.

Anthelmintic drug residues in beef: UPLC-MS/MS method validation, European retail beef survey, and associated exposure and risk assessments

Anthelmintic drugs are widely used to control parasitic infections in cattle. The ProSafeBeef project addressed the need for data on the exposure of European consumers of beef to potentially harmful drug residues. A novel analytical method based on matrix solid-phase dispersive extraction and ultra-performance liquid chromatography-tandem mass spectrometry was validated for 37 anthelmintic drugs and metabolites in muscle (assay decision limits, CCa, = 0.15-10.2 µg kg -1). Seven European countries (France, Spain, Slovenia, Ireland, Italy, Belgium and Portugal) participated in a survey of retail beef purchased in local shops. Of 1061 beef samples analysed, 26 (2.45%) contained detectable residues of anthelmintic drugs (0.2-171 µg kg -1), none above its European Union maximum residue limit (MRL) or action level. Residues detected included closantel, levamisole, doramectin, eprinomectin, moxidectin, ivermectin, albendazole and rafoxanide. In a risk assessment applied to mean residue concentrations across all samples, observed residues accounted for less than 0.1% of the MRL for each compound. An exposure assessment based on the consumption of meat at the 99th percentile of consumption of adults in 14 European countries demonstrated that beef accounted for less than 0.02% of the acceptable daily intake for each compound in each country. This study is the first of its kind to apply such a risk-based approach to an extensive multi-residue survey of veterinary drug residues in food. It has demonstrated that the risk of exposure of the European consumer to anthelmintic drug residues in beef is negligible, indicating that regulation and monitoring is having the desired effect of limiting residues to non-hazardous concentrations. © 2012 Copyright Taylor and Francis Group, LLC.

Origin of double dinitrogen release feature during fast switching between lean and rich cycles for NOx storage reduction catalysts

The performance of NOx storage and reduction over 1.5 wt% Pt/20 wt% KNO3/K2Ti8O17 and 1.5 wt% Pt/K2Ti8O17 catalysts has been investigated using combined fast transient kinetic switching and isotopically labelled (NO)-N-15 at 350 degrees C. The evolution of product N-2 has revealed two significant peaks during 60 s lean/1.3 s rich switches. It also found that the presence of CO2 in the feed affects the release of N-2 in the second peak. Regardless of the presence/absence of water in the feed, only one peak of N-2 was observed in the absence of CO2. Gas-phase NH3 was not observed in any of the experiments. However, in the presence of CO2 the results obtained from in situ DRIFTS-MS analysis showed that isocyanate species are formed and stored during the rich cycles, probably from the reaction between NOx and CO, in which CO was formed via the reverse water-gas shift reaction. 

Linking watershed terrain and hydrology to soil chemical properties, microbial communities and impacts on soil organic C in a humid mid-latitude forested watershed

Understanding the response of humid mid-latitude forests to changes in precipitation, temperature, nutrient cycling, and disturbance is critical to improving our predictive understanding of changes in the surface-subsurface energy balance due to climate change. Mechanistic understanding of the effects of long-term and transient moisture conditions are needed to quantify
linkages between changing redox conditions, microbial activity, and soil mineral and nutrient interactions on C cycling and greenhouse gas releases. To illuminate relationships between the soil chemistry, microbial communities and organic C we established transects across hydraulic and topographic gradients in a small watershed with transient moisture conditions. Valley bottoms tend to be more frequently saturated than ridge tops and side slopes which generally are only saturated when shallow storm flow zones are active. Fifty shallow (~36”) soil cores were collected during timeframes representative of low CO2, soil winter conditions and high CO2, soil summer conditions. Cores were subdivided into 240 samples based on pedology and analyses of the geochemical (moisture content, metals, pH, Fe species, N, C, CEC, AEC) and microbial (16S rRNA gene
amplification with Illumina MiSeq sequencing) characteristics were conducted and correlated to watershed terrain and hydrology. To associate microbial metabolic activity with greenhouse gas emissions we installed 17 soil gas probes, collected gas samples for 16 months and analyzed them for CO2 and other fixed and greenhouse gasses. Parallel to the experimental efforts our data is being used to support hydrobiogeochemical process modeling by coupling the Community Land Model (CLM) with a subsurface process model (PFLOTRAN) to simulate processes and interactions from the molecular to watershed scales. Including above ground processes (biogeophysics, hydrology, and vegetation dynamics), CLM provides mechanistic water, energy, and organic matter inputs to the surface/subsurface models, in which coupled biogeochemical reaction
networks are used to improve the representation of below-ground processes. Preliminary results suggest that inclusion of above ground processes from CLM greatly improves the prediction of moisture response and water cycle at the watershed scale.

pLR-HL: a novel amphibian Bowman-Birk-type trypsin inhibitor from the skin secretion of the broad-folded frog, Hylarana latouchii

In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a "shotgun" cloning technique. It contains a disulphide loop between Cys5 and Cys15 which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesised by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nM. The substitution of Lys-8 by Phe (Phe8 -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μM. Additionally, both the disulphide loops of pLR-HL and Phe8 -pLR-HL were synthesised and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically-active moiety.

Biomass-derived oxymethylene ethers as diesel additives: A techno-economic assessment

Conversion of agricultural biomass such as wood chips, wheat straw and forest residue for the production of fuels can help in reducing GHG emissions since they are considered as nearly carbon neutral. Around the world there is a significant amount of forest and agricultural-biomass available which could be used for the production of liquid fuels that can be blended with the petroleum-based diesel. Oxymethylene ethers (OMEs) can be derived from biomass via gasification, water-gas shift reaction and methanol production. The addition of OMEs to conventional diesel fuel has great potential to reduce soot formation during the combustion in diesel engines. Unlike methanol and dimethyl ether (DMM) which can also reduce soot formation, the physical properties of OMEs allow the use in modern diesel engines without significant change of the engines infrastructure. In this study, a detailed and data intensive process simulation model was developed to simulate all the unit operations involved in the production of OMEs from biomass. The unit operation considered include biomass drying, gasification, gas cleaning, water gas shift reaction, methanol production and OMEs synthesis. The simulation results were then utilized to conduct a detailed techno-economic assessment study of the whole biomass conversion chain to determine the most attractive pathways for OMEs production. Our recent study shows that the key parameters affecting the OMEs production are equivalence ratio, H2/CO ratio and optimal air flow. Overall, the cost of production ($/liter) of OMEs from different biomass feedstock in Alberta will be determined

What defines a geochemical map? Implications for environmental assessment.

The environmental quality of land is often assessed by the calculation of threshold values which aim to differentiate between concentrations of elements based on whether the soils are in residential or industrial sites. In Europe, for example, soil guideline values exist for agricultural and grazing land. A threshold is often set to differentiate between concentrations of the element that naturally occur in the soil and concentrations that result from diffuse anthropogenic sources. Regional geochemistry and, in particular, single component geochemical maps are increasingly being used to determine these baseline environmental assessments. The key question raised in this paper is whether the geochemical map can provide an accurate interpretation on its own. Implicit is the thought that single component geochemical maps represent absolute abundances. However,because of the compositional (closed) nature of the data univariate geochemical maps cannot be compared directly with one another.. As a result, any interpretation based on them is vulnerable to spurious correlation problems. What does this mean for soil geochemistry mapping, baseline quality documentation, soil resource assessment or risk evaluation? Despite the limitation of relative abundances, individual raw geochemical maps are deemed fundamental to several applications of geochemical maps including environmental assessments. However, element toxicity is related to its bioavailable concentration, which is lowered if its source is mixed with another source. Elements interact, for example under reducing conditions with iron oxides, its solid state is lost and arsenic becomes soluble and mobile. Both of these matters may be more adequately dealt with if a single component map is not interpreted in isolation to determine baseline and threshold assessments. A range of alternative compositionally compliant representations based on log-ratio and log-contrast approaches are explored to supplement the classical single component maps for environmental assessment. Case study examples are shown based on the Tellus soil geochemical dataset, covering Northern Ireland and the results of in vitro oral bioaccessibility testing carried out on a sub-set of archived Tellus Survey shallow soils following the Unified BARGE (Bioaccessibility Research Group of Europe).

Laser-driven x-ray and neutron source development for industrial applications of plasma accelerators

Pulsed beams of energetic x-rays and neutrons from intense laser interactions with solid foils are promising for applications where bright, small emission area sources, capable of multi-modal delivery are ideal. Possible end users of laser-driven multi-modal sources are those requiring advanced non-destructive inspection techniques in industry sectors of high value commerce such as aerospace, nuclear and advanced manufacturing. We report on experimental work that demonstrates multi-modal operation of high power laser-solid interactions for neutron and x-ray beam generation. Measurements and Monte Carlo radiation transport simulations show that neutron yield is increased by a factor ∼2 when a 1 mm copper foil is placed behind a 2 mm lithium foil, compared to using a 2 cm block of lithium only. We explore x-ray generation with a 10 picosecond drive pulse in order to tailor the spectral content for radiography with medium density alloy metals. The impact of using >1 ps pulse duration on laser-accelerated electron beam generation and transport is discussed alongside the optimisation of subsequent bremsstrahlung emission in thin, high atomic number target foils. X-ray spectra are deconvolved from spectrometer measurements and simulation data generated using the GEANT4 Monte Carlo code. We also demonstrate the unique capability of laser-driven x-rays in being able to deliver single pulse high spatial resolution projection imaging of thick metallic objects. Active detector radiographic imaging of industrially relevant sample objects with a 10 ps drive pulse is presented for the first time, demonstrating that features of 200 μm size are resolved when projected at high magnification.

CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF ESSENTIAL OILS OF LAVANDULA SPP.

Lavenders belong to the family Labiatae and represent some of the most popular medicinal plants of great economic importance. Their essential oils are important for the perfume, cosmetic, flavouring and pharmaceutical industries. However, despite its popularity, and the long tradition of use, biological properties of the various Lavandula species are not yet been well sustained by scientific or clinical studies and some available data being inconclusive and controversial [1]. Although Lavandula spp. have similar ethnobotanical properties, however, chemical composition and therapeutic uses differ from different species and main composition of essential oils showed differences with species and with the region were they grow [1,2,3]. L. stoechas L. subsps. luisieri (Rozeira) Rozeira. L. pedunculata (Mill.) Cav. and L. viridis L’Hér are endemic to the Iberian Peninsula, widespread in the South of Portugal, namely in Alentejo and Algarve. In our work, essential oils from the stems or leaves from wild grown plants of L. luisieri (Alentejo), L. pedunculata (Alentejo) and L. viridis (Algarve), were extracted by hydrodistillation and analyzed by GC-FID. Antimicrobial activity was evaluated by solid diffusion disk assay and minimal inhibitory concentration (MIC) against pathogenic Gram-positive and Gram-negative bacteria and food spoilage fungi.

Novos sistemas catalíticos baseados em óxidos de metais de transição

A presente dissertação descreve a síntese de novos sistemas catalíticos: (a) os complexos MoO2Cl2(pypzH), [MoO2Cl2(pypzH)]·pyHpzHCl, Mo2O4(μ2- O)Cl2(pypzH)2, MoO(O2)2Cl(pyHpzH), MoO2(OSiPh3)2(pypzH) (pypzH = 2-[3(5)- pirazolil]piridina), (η6-C6H5CH3)Mo(CO)3, (b) os materiais híbridos orgânicosinorgânicos PMO-ph suportados com as unidades Mo(CO)3 e Cr(CO)3, (c) e os complexos de inclusão CpMo(CO)3Cl@β-CD e CpMo(CO)3Cl@TRIMEB. Os novos sistemas catalíticos foram caracterizados utilizando os métodos de caracterização usuais (AE, FT-IR, Raman, NMR 1H) assim como os métodos de caracterização de estado sólido (XRD de pós, de cristal único, 13C e 29Si NMR CP MAS) e pela técnica microscópica de alta resolução (TEM). Os compostos com propriedades catalíticas e pré-catalíticas promissoras foram investigados nas reacções catalíticas de epoxidação de várias olefinas, nomeadamente o cis-cicloocteno.

Maturação de lamas vulcânicas açorianas e sua aplicação em peloterapia

Em Portugal, nomeadamente nos Açores (Centro Termal das Furnas), durante vários anos foram utilizados pelóides naturais (lamas/argilas obtidos após conveniente maturação com água mineral) como agentes terapêuticos. Apesar das evidências empíricas da aplicação das argilas para fins terapêuticos, o suporte científico é ainda escasso. Além disso, contrariamente ao que se verifica para águas minerais utilizadas para fins termais, no campo da peloterapia, não existe regulamentação que estabeleça critérios quanto à qualidade padrão de um pelóide. Este trabalho visou a avaliação das características biológicas, físico-químicas e mineralógicas de lamas vulcânicas das ilhas de São Miguel e da Terceira para aplicação em peloterapia antes e após maturação. A área de amostragem foi caracterizada sob o ponto de vista geomorfológico, geológico, hidrológico e climático. A análise dos materiais colhidos contemplou estudos de granulometria e de mineralogia da amostra total e da fracção argilosa; caracterização química; e determinação de outras propriedades tais como índice de abrasividade, superfície específica, pH, capacidade de troca catiónica e catiões de troca, limites de consistência (plasticidade e liquidez e índice de plasticidade) e taxa de arrefecimento. A caracterização biológica das lamas incluiu a identificação e quantificação relativa de diatomáceas e a determinação do teor em coliformes totais e microrganismos cultiváveis a 37 ºC e a 22 ºC. Estudos efectuados em amostras comerciais argilosas e em recursos naturais açorianos precederam os ensaios de maturação. Estes estudos visaram, fundamentalmente, aferir as características apropriadas dos materiais para aplicação tópica e detectar e caracterizar a presença de comunidades diatomológicas em locais próximos dos materiais a serem sujeitos a maturação. O estudo de maturação foi desenvolvido em duas etapas sequenciais e complementares com vista à optimização do processo de maturação de lamas vulcânicas açorianas. Foi testada a influência de algumas condições abióticas na maturação e fez-se variar a fracção granulométrica, proporção fase sólida:fase líquida e o tempo de maturação entre os dois ensaios. O uso da granulometria <63 μm, proporção de 2:1 (fase sólida:fase líquida) e condições de luminosidade, sem agitação parecem ser as mais adequadas à maturação dos materiais analisados. A maturação revela-se um processo que promove a melhoria de características importantes dos materiais para aplicação em peloterapia, tais como aumento dos tempos de arrefecimento e da quantidade de catiões de troca. Existe uma comunidade de microalgas e bacteriana capaz de se desenvolver em materiais geológicos vulcânicos durante a maturação e cujo contributo para a acção terapêutica do pelóide deverá ser aferida. Após comparação com materiais de referência, a amostra das Furnas revela as características mais adequadas para aplicação em peloterapia.

Dinâmicas territoriais geradas pelo investimento privado no turismo

A importância económica, social e política do setor do turismo é uma realidade inquestionável. A sua expressão e capacidade de induzir o desenvolvimento económico em termos de outros setores, é cada vez mais importante. Apesar da dinâmica demonstrada, o setor do turismo enfrenta uma série de problemas estruturais que devem ser resolvidos. Nos últimos anos, o turismo em Portugal tem vindo a crescer em quantidade, mas às vezes sem muita qualidade. O desempenho económico do setor deve ser devidamente analisado, desde a análise do número de entradas de visitantes até questões como o volume de receitas obtido. Para além disso, é possível observar a existência de casos de concentração espacial excessiva em certas áreas do território. A política de turismo precisa de dimensão, de ser forte, bem estruturada e orientada. "Esta política deve promover sinergias entre redes de diferentes atores para criar uma base sólida de segurança e confiança para o investimento privado." (Costa, 2000) É importante avaliar o investimento e o sistema de financiamento no setor do turismo, para entender a filosofia e os objetivos que norteiam todo o sistema (política estratégica) e as dinâmicas territoriais e setoriais das empresas do turismo.

Severe plastic deformation of Al–Zn alloys

In this work, the R&D work mainly focused on the mechanical and microstructural analysis of severe plastic deformation (SPD) of Al–Zn alloys and the development of microstructure–based models to explain the observed behaviors is presented. Evolution of the microstructure and mechanical properties of Al–30wt% Zn alloy after the SPD by the high–pressure torsion (HPT) has been investigated in detail regarding the increasing amount of deformation. SPD leads to the gradual grain refinement and decomposition of the Al–based supersaturated solid solution. The initial microstructure of the Al–30wt% Zn alloy contains Al and Zn phases with grains sizes respectively of 15 and 1 micron. The SPD in compression leads to a gradual decrease of the Al and Zn phase grain sizes down to 4 microns and 252 nm, respectively, until a plastic strain of 0.25 is reached. At the same time, the average size of the Zn particles in the bulk of the Al grains increases from 20 to 60 nm and that of the Zn precipitates near or at the grain boundaries increases as well. This microstructure transformation is accompanied at the macroscopic scale by a marked softening of the alloy. The SPD produced by HPT is conducted up to a shear strain of 314. The final Al and Zn grains refine down to the nanoscale with sizes of 370 nm and 170 nm, respectively. As a result of HPT, the Zn–rich (Al) supersaturated solid solution decomposes completely and reaches the equilibrium state corresponding to room temperature and its leads to the material softening. A new microstructure–based model is proposed to describe the softening process occurring during the compression of the supersaturated Al–30wt% Zn alloy. The model successfully describes the above–mentioned phenomena based on a new evolution law expressing the dislocation mean free path as a function of the plastic strain. The softening of the material behavior during HPT process is captured very well by the proposed model that takes into consideration the effects of solid solution hardening and its decomposition, Orowan looping and dislocation density evolution. In particular, it is demonstrated that the softening process that occurs during HPT can be attributed mainly to the decomposition of the supersaturated solid solution and, in a lesser extent, to the evolution of the dislocation mean free path with plastic strain.

Development of a simple analytical method for the simultaneousdetermination of paracetamol, paracetamol-glucuronide andp-aminophenol in river water

Paracetamol is among the most worldwide consumed pharmaceuticals. Although its occurrence in the environment is well documented, data about the presence of its metabolites and transformation products is very scarce. The present work describes the development of an analytical method for the simultaneous determination of paracetamol, its principal metabolite (paracetamol-glucuronide) and its main transformation product (p-aminophenol) based on solid phase extraction (SPE) and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). The method was applied to analysis of river waters, showing to be suitable to be used in routine analysis. Different SPE sorbents were compared and the use of two Oasis WAX cartridges in tandem proved to be the most adequate approach for sample clean up and pre-concentration. Under optimized conditions, limits of detection in the range 40–67 ng/L were obtained, as well as mean recoveries between 60 and 110% with relative standard deviations (RSD) below 6%. Finally, the developed SPE-HPLC/DAD method was successfully applied to the analysis of the selected compounds in samples from seven rivers located in the north of Portugal. Nevertheless all the compounds were detected, it was the first time that paracetamol-glucuronide was found in river water at concentrations up to 3.57 μg/L.