Enrichment of pathogenic alleles in the brittle cornea gene, ZNF469, in keratoconus

Keratoconus, a common inherited ocular disorder resulting in progressive corneal thinning, is the leading indication for corneal transplantation in the developed world. Genome-wide association studies have identified common SNPs 100 kb upstream of ZNF469 strongly associated with corneal thickness. Homozygous mutations in ZNF469 and PR domain-containing protein 5 (PRDM5) genes result in brittle cornea syndrome (BCS) Types 1 and 2, respectively. BCS is an autosomal recessive generalized connective tissue disorder associated with extreme corneal thinning and a high risk of corneal rupture. Some individuals with heterozygous PRDM5 mutations demonstrate a carrier ocular phenotype, which includes a mildly reduced corneal thickness, keratoconus and blue sclera. We hypothesized that heterozygous variants in PRDM5 and ZNF469 predispose to the development of isolated keratoconus. We found a significant enrichment of potentially pathologic heterozygous alleles in ZNF469 associated with the development of keratoconus (P = 0.00102) resulting in a relative risk of 12.0. This enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients represents a significant mutational load and highlights ZNF469 as the most significant genetic factor responsible for keratoconus identified to date.

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.

Evaluating environmental enrichment options for commercial broiler chickens

Implications Provision of environmental enrichment in line with that required by welfare-based quality assurance schemesdoes not always appear to lead to clear improvements in broiler chicken welfare. This research perhaps serves to highlightthe deficit in information regarding the ‘real world’ implications of enrichment with perches, string and straw bales.

Introduction Earlier work showed that provision of natural light and straw bales improved leg health in commercial broilerchickens (Bailie et al., 2013). This research aimed to determine if additional welfare benefits were shown in windowedhouses by increasing straw bale provision (Study 1), or by providing perches and string in addition to straw bales (Study 2).

Material and methods Commercial windowed houses in Northern Ireland containing ~23,000 broiler chickens (placed inhouses as hatched) were used in this research which took place in 2011. In Study 1 two houses on a single farm wereassigned to one of two treatments: (1) 30 straw bales per house (1 bale/44m2), or (2) 45 straw bales per house (1bale/29m2). Bales of wheat straw, each measuring 80cm x 40cm x 40cm were provided from day 10 of the rearing cycle,as in Bailie et al. (2013). Treatments were replicated over 6 production cycles (using 276,000 Ross 308 and Cobb birds),and were swapped between houses in each replicate. In Study 2, four houses on a single farm were assigned to 1 of 4treatments in a 2 x 2 factorial design. Treatments involved 2 levels of access to perches (present (24/house), or absent), and2 levels of access to string (present (24/house), or absent), and both types of enrichment were provided from the start of thecycle. Each perch consisted of a horizontal, wooden beam (300 cm x 5 cm x 5cm) with a rounded upper edge resting on 2supports (15 cm high). In the string treatment, 6 pieces of white nylon string (60 cm x 10 mm) were tied at their mid-pointto the wire above each of 4 feeder lines. Thirty straw bales were also provided per house from day 10. This study wasreplicated over 4 production cycles using 368,000 Ross 308 birds. In both studies behaviour was observed between 0900and 1800 hours in weeks 3-5 of the cycle. In Study 1, 8 focal birds were selected in each house each week, and generalactivity, exploratory and social behaviours recorded directly for 10 minutes. In Study 2, 10 minute video recordings weremade of 6 different areas (that did not contain enrichment) of each house each week. The percentage of birds engaged inlocomotion or standing was determined through scan sampling these recordings at 120 second intervals. Four perches andfour pieces of string were filmed for 25 mins in each house that contained these enrichments on one day per week. The totalnumber of times the perch or string was used was recorded, along with the duration of each bout. In both studies, gaitscores (0 (perfect) to 5 (unable to walk)) and latency to lie (measured in seconds from when a bird had been encouraged tostand) were recorded in 25 birds in each house each week. Farm and abattoir records were also used in both studies todetermine the number of birds culled for leg and other problems, mortality levels, slaughter weights, and levels of pododermatitis and hock burn. Data were analysed using SPSS (version 20.0) and treatment and age effects on behaviouralparameters were determined in normally distributed data using ANOVA (‘Straw bale density*week’, or‘string*perches*week’ as appropriate), and in non-normally distributed data using Kuskall-Wallace tests (P<0.05 forsignificance) . Treatment (but not age) effects on performance and health data were determined using the same testsdepending on normality of data.

Results Average slaughter weight, and levels of mortality, culling, hock burn and pododermatitis were not affected bytreatment in either study (P<0.05). In Study 1 straw bale (SB) density had no significant effect on the frequency orduration of behaviours including standing, walking, ground pecking, dust bathing, pecking at bales or aggression, or onaverage gait score (P>0.05). However, the average latency to lie was greater when fewer SB were provided (30SB 23.38s,45SB 18.62s, P<0.01). In Study 2 there was an interaction between perches (Pe) and age in lying behaviour, with higherpercentages of birds observed lying in the Pe treatment during weeks 4 and 5 (week 3 +Pe 77.0 -Pe 80.9, week 4 +Pe 79.5 -Pe 75.2, week 5 +Pe 78.4 -Pe 76.2, P<0.02). There was also a significant interaction between string (S) and age inlocomotory behaviour, with higher percentages of birds observed in locomotion in the string treatment during week 3 butnot weeks 4 and 5 (week 3 +S 4.9 -S 3.9, week 4 +S 3.3 -S 3.7, week 5 +S 2.6 -S 2.8, P<0.04). There was also aninteraction between S and age in average gait scores, with lower gait scores in the string treatment in weeks 3 and 5 (week3: +S 0.7, -S 0.9, week 4: +S 1.5, -S 1.4, week 5: +S 1.9, -S 2.0, P<0.05). On average per 25 min observation there were15.1 (±13.6) bouts of perching and 19.2 (±14.08) bouts of string pecking, lasting 117.4 (±92.7) and 4.2 (±2.0) s for perchesand string, respectively.

Conclusion Increasing straw bale levels from 1 bale/44m2 to 1 bale/29m2 floor space does not appear to lead to significantimprovements in the welfare of broilers in windowed houses. The frequent use of perches and string suggests that thesestimuli have the potential to improve welfare. Provision of string also appeared to positively influence walking ability.However, this effect was numerically small, was only shown in certain weeks and was not reflected in the latency to lie.Further research on optimum design and level of provision of enrichment items for broiler chickens is warranted. Thisshould include measures of overall levels of activity (both in the vicinity of, and away from, enrichment items).

A comparative study on the thermophysical properties for two bis[(trifluoromethyl)sulfonyl]imide-based ionic liquids containing the trimethyl-sulfonium or the trimethyl-ammonium cation in molecular solvents

Herein, we present a comparative study of the thermophysical properties of two homologous ionic liquids, namely, trimethyl-sulfonium bis[(trifluoromethyl) sulfonyl]imide, [S₁₁₁][TFSI], and trimethyl-ammonium bis[(trifluoromethyl)sulfonyl]imide, [HN₁₁₁][TFSI], and their mixtures with propylene carbonate, acetonitrile, or gamma butyrolactone as a function of temperature and composition. The influence of solvent addition on the viscosity, conductivity, and thermal properties of IL solutions was studied as a function of the solvent mole fraction from the maximum solubility of IL, x_s, in each solvent to the pure solvent. In this case, x_s is the composition corresponding to the maximum salt solubility in each liquid solvent at a given temperature from 258.15 to 353.15 K. The effect of temperature on the transport properties of each binary mixture was then investigated by fitting the experimental data using Arrhenius' law and the Vogel-Tamman-Fulcher (VTF) equation. The experimental data shows that the residual conductivity at low temperature, e.g., 263.15 K, of each binary mixture is exceptionally high. For example, conductivity values up to 35 and 42 mS·cm^-1 were observed in the case of the [S ₁₁₁][TFSI] + ACN and [HN₁₁₁][TFSI] + ACN binary mixtures, respectively. Subsequently, a theoretical approach based on the conductivity and on the viscosity of electrolytes was formulated by treating the migration of ions as a dynamical process governed by ion-ion and solvent-ion interactions. Within this model, viscosity data sets were first analyzed using the Jones-Dole equation. Using this theoretical approach, excellent agreement was obtained between the experimental and calculated conductivities for the binary mixtures investigated at 298.15 K as a function of the composition up to the maximum solubility of the IL. Finally, the thermal characterization of the IL solutions, using DSC measurements, showed a number of features corresponding to different solid-solid phase transitions, T_S-S, with extremely low melting entropies, indicating a strong organizational structure by easy rotation of methyl group. These ILs can be classified as plastic crystal materials and are promising as ambient-temperature solid electrolytes. © 2013 American Chemical Society.

A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate

Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.

High temperature thermal stability investigations of ammonium sulphide passivated InGaAs and interface formation with Al2O3 studied by synchrotron radiation based photoemission

High resolution synchrotron radiation core level photoemission measurements have been used to undertake a comparative study ofthe high temperature thermal stability ofthe ammonium sulphide passivated InGaAs surface and the same surface following the atomic layer deposition (ALD) of an ultrathin (∼1 nm) Al2O3 layer. The solution based ex situ sulphur passivation was found to be effective at removing a significant amount of the native oxides and protecting the surface against re-oxidation upon air exposure. The residual interfacial oxides which form between sulphur passivated InGaAs and the ultrathin Al2O3 layer can be substantially removed at high temperature (up to 700 ◦C) without impacting on the InGaAs stoichiometry while significant loss of indium was recorded at this temperature on the uncovered sulphur passivated InGaAs surface.

Tracing coastal organic enrichment: stable isotopes and biotic indices

Neste trabalho foi efectuada uma avaliação integrada usando descritores sedimentares e biológicos ao nível da espécie e da comunidade e índices bióticos de síntese para o traçamento do enriquecimento orgânico numa região, com características dispersivas, da costa Oeste de Portugal. Na área estudada existem gradientes ambientais e biológicos relacionados com a heterogeneidade da paisagem sedimentar, a qual inclui sedimentos desde areias finas limpas a vasas. Contudo, na área próxima do emissário, esta paisagem é mais homogénea e constituída por areia fina com baixo teor em finos. Nesta região, alguns dos descritores estudados deram uma indicação coerente de alterações ambientais associadas ao enriquecimento orgânico. O potencial de oxidação - redução mostrou valores negativos até 250 m do emissário, o que indicia que a degradação da matéria orgânica que entra no sistema cria condições reduzidas no sedimento. Os isótopos estáveis de carbono e azoto no sedimento diferenciam a área mais próxima do emissário, que apresenta uma depleção de acordo com uma origem terrestre da matéria orgânica naquela parte da plataforma. Uma imagem similar foi obtida pela análise dos isótopos estáveis na macrofauna que diagnosticou a origem terrestre da matéria orgânica consumida. A composição específica e a abundância das comunidades bentónicas também são significativamente diferentes junto ao emissário, onde são dominadas por espécies oportunistas, tolerantes ao enriquecimento orgânico. No entanto, os índices bióticos em validação no âmbito da implementação da Directiva Quadro da Água, não foram eficientes a mostrar as alterações bentónicas associadas ao enriquecimento orgânico apesar de alguns índices se basearem nos limiares de tolerância/sensibilidade a este tipo de perturbação. Apesar deste caso de estudo reflectir um enriquecimento orgânico moderado, uma vez que não foram detectadas alterações sedimentares ou acumulação de matéria orgânica, nem um significativo empobrecimento das comunidades biológicas junto ao emissário, a análise ao nível dos índices bióticos de síntese pode levar à perda de informação essencial e, portanto, prejudicar a nossa capacidade de diagnóstico devendo ser usados com cuidado. A análise do conjunto de dados da composição específica forneceu uma imagem mais precisa da perturbação ambiental e descritores específicos, tais como os isótopos estáveis, permitiram uma melhor compreensão da extensão espacial do enriquecimento orgânico.

Primary production in systems subject to natural and anthropogenic eutrophication

Tese dout., Química, Universidade do Algarve, 2005

Bottom-up regulation of phytoplankton in the Guadiana estuary

Tese dout., Ciências do Mar (Ecologia Marinha), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Effects of nutrient and light enrichment on phytoplankton growth

Alterations of freshwater flow regimes and increasing eutrophication lead to alterations in light availability and nutrient loading into adjacent estuaries and coastal areas. Phytoplankton community respond to these changes in many ways. Harmful phytoplankton blooms, for instance, may be a consequence of changes in nutrient supply, as well as the replacement of some phytoplankton species (like diatoms, that contribute for the development of large fish and shellfish populations) by ohers (like cyanobacteria, that may be toxic and represent an undesirable food source for higher trophic levels). Nutrient and light enrichment experiments allow us to understand and predict the effects of eutrophication on the growth of phytoplankton. This is a fundamental tool in water management issues, since it enables the prediction of changes in the phytoplankton community that may be harmful to the whole ecosystem, and the design of mitigation strategies (Zalewski 2000).

RT-Bst: an integrated approach for reverse transcription and enrichment of cDNA from viral RNA

The synthesis of cDNA from RNA is challenging due to the inefficiency of reverse transcription (RT). In order to address this, a method was developed known as RT-Bst for sequential RT of RNA and Bst DNA polymerase amplification for enrichment of cDNA in a single tube reaction. Using genomic RNA from bacteriophage MS2, the yield of cDNA produced by RT alone and RT-Bst were compared by analysis of PCR-amplified products. Using random primers a superior performance was observed when amplifying MS2 RNA following RT-Bst compared to RT alone, indicating that greater quantities of cDNA were present after RT-Bst. RT-Bst was also compared with RT alone for their relative ability to produce sufficient cDNA to amplify 8 target regions spanning the respiratory syncytial virus (RSV) genome. Six out of 8 targets were amplified consistently by PCR subsequent to RT-Bst amplification whereas only 3 out of 8 targets could be amplified after RT alone. RSV sequences were selectively amplified using RSV specific primers from a mixed template containing an excess of MS2 RNA in a RT-Bst reaction without amplifying MS2 sequences. This suggests that RT-Bst can be used to amplify RNA sequences non-specifically using random primers and specifically using sequence specific primers and enhances the yield of cDNA when compared to RT alone.

Enrichment of bioactive compounds in microalgae for aquaculture

Microalgae are promising microorganisms for the production of food and fine chemicals. Several species of microalgae are used in aquaculture with the purpose of transfer bioactive compounds up to the aquatic food chain. The main objective of this project was to develop a stress–inducement strategy in order to enhance the biochemical productivity of Nannochloropsis gaditana, Rhodomonas marina and Isochrysis sp. for aquaculture purposes having in account their growth and organizational differences. In this regard, two experiments were design: the first one consisted on the alteration of overall nutrient availabilities in growth medium; and the second one comprised changes in nitrogen and sulfur concentrations maintaining the concentrations of the other nutrients present in a commercial growth medium (Nutribloom plus), which is frequently used in aquaculture. Microalgae dried biomass was characterized biochemically and elemental analysis was also performed for all samples. In first experimental design: linear trends between nutrient availability in growth media and microalgae protein content were obtained; optimum productivities of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) were attained for both R. marina and N. gaditana in growth media enriched with 1000 L L-1 of nutrient solution whereas for Isochrysis sp. the double of Nutribloom plus was needed; the decrease of glucans and total monosaccharides with nutrient availability for R. marina and Isochrysis sp. showed the occurrence of a possible depletion of carbohydrates towards lipids and proteins biosynthesis. Second experimental desing: N. gaditana exhibited the highest variation in their biochemical composition against the applied perturbation; variations observed for microalgae in their biochemical composition were reflected in their elemental stoichiometry; in N. gaditana the highest nitrogen concentrations lead to overall maximum productivities of the biochemical parameters. The results of the present work show two stress-inducement strategies for microalgae that may constitute a base for further investigations on their biochemical enhancement.