Heteroduplex formation and S1 digestion for mapping alternative splicing sites

The identification of alternatively spliced transcripts has contributed to a better comprehension of developmental mechanisms, tissue-specific physiological processes and human diseases. Polymerase chain reaction amplification of alternatively spliced variants commonly leads to the formation of heteroduplexes as a result of base pairing involving exons common between the two variants. S1 nuclease cleaves single-stranded loops of heteroduplexes and also nicks the opposite DNA strand. In order to establish a strategy for mapping alternative splice-prone sites in the whole transcriptome, we developed a method combining the formation of heteroduplexes between 2 distinct splicing variants and S1 nuclease digestion. For 20 consensuses identified here using this methodology, 5 revealed a conserved splice site after inspection of the cDNA alignment against the human genome (exact splice sites). For 8 other consensuses, conserved splice sites were mapped at 2 to 30 bp from the border, called proximal splice sites; for the other 7 consensuses, conserved splice sites were mapped at 40 to 800 bp, called distal splice sites. These latter cases showed a nonspecific activity of S1 nuclease in digesting double-strand DNA. From the 20 consensuses identified here, 5 were selected for reverse transcription-polymerase chain reaction validation, confirming the splice sites. These data showed the potential of the strategy in mapping splice sites. However, the lack of specificity of the S1 nuclease enzyme is a significant obstacle that impedes the use of this strategy in large-scale studies.

Partial microwave-assisted wet digestion of animal tissue using a baby-bottle sterilizer for analyte determination by inductively coupled plasma optical emission spectrometry

A procedure for partial digestion of bovine tissue is proposed using polytetrafluoroethylene (PTFE) microvessels inside a baby-bottle sterilizer under microwave radiation for multi-element determination by inductively coupled plasma optical emission spectrometry (ICP OES). Samples were directly weighed in laboratory-made polytetrafluoroethylene vessels. Nitric acid and hydrogen peroxide were added to the uncovered vessels, which were positioned inside the baby-bottle sterilizer, containing 500 mL of water. The hydrogen peroxide volume was fixed at 100 mu L The system was placed in a domestic microwave oven and partial digestion was carried out for the determination of Ca, Cu, Fe. Mg, Mn and Zn by inductively coupled plasma optical emission spectrometry. The single-vessel approach was used in the entire procedure, to minimize contamination in trace analysis. Better recoveries and lower residual carbon content (RCC) levels were obtained under the conditions established through a 2(4-1) fractional factorial design: 650 W microwave power, 7 min digestion time, 50 mu L nitric acid and 50 mg sample mass. The digestion efficiency was ascertained according to the residual carbon content determined by inductively coupled plasma optical emission spectrometry. The accuracy of the proposed procedure was checked against two certified reference materials. (C) 2009 Elsevier B.V. All rights reserved.

Sheep rumen digestion and transmission of weedy Malva parviflora seeds

The effect of sheep digestion and mastication on Malva parviflora L. seed transmission, viability and germination was investigated. Mature M. parviflora seeds were subjected to 2 seed treatments: 'scarified', where the hard seed coat was manually cut to allow inhibition, and 'unscarified', where the hard seed coat was not cut. Seeds were placed directly into the rumen of fistulated sheep and removed at 0, 12, 24, 36 and 48 h of rumen digestion. After 12 h of in sacco exposure to digestion in the rumen, the germination of seeds that were initially scarified dropped from 99.2 to 1.4% and longer exposure periods produced no germinable seeds. In contrast, seeds that were unscarified when placed in the rumen produced over 92% germination regardless of in sacco digestion time, although manual scarification after retrieval was essential to elicit germination. In a second experiment, unscarified seeds (29000) were fed in a single meal to fistulated sheep and feces were collected at regular intervals between 6 and 120 h after feeding. Fecal subsamples were taken to determine number of seeds excreted, seed germination on agar and seed germination from feces. Major seed excretion in the feces commenced after 12 h and continued until 144 h, with peaks between 36 and 72 h after consumption. Although mastication and gut passage killed the majority of unscarified seeds, about 20% were recovered intact and over 90% of these recovered seeds were viable and could, thus, potentially form an extensive seed bank. A few excreted seeds (1%) were able to germinate directly from feces, which increased to a maximum of 10% after subsequent dry summer storage (3 months). Through information gained in this study, there is a potential to utilise livestock in an integrated weed management program for the control of M. parviflora, provided additional measures of weed control are in place such as holding periods (> 7 days) for movement of livestock from weed infested areas.

Productive performance, nutrient digestion and metabolism of Holstein (Bos taurus) and Nellore (Bos taurus indicus) cattle and Mediterranean Buffaloes (Bubalis bubalis) fed with corn-silage based diets

The objective of this study was to evaluate the productive performance, nutrients digestion and metabolism of three different genetic groups fed with the same diet based on corn silage. 30 heifers in growth were used of three groups of cattle, the following: Nellore (Bos taurus indicus) (n = 10), Holstein (Bos taurus taurus) (n = 10), and Mediterranean buffaloes (Bubalis bubalis) (n = 10). The animals were fed in groups and received the same experimental diet composed of corn silage and concentrate for growing heifers. In the evaluation of animals the performance, consumption and total apparent digestibility of dry matter and nutrients with the aid of internal markers (chromic oxide) and external (iADF), rumen fermentation, excretion of purine derivatives, nitrogen balance and blood metabolites were measured. No differences were observed in animal performance. There were differences in nutrient intake and apparent digestibility of dry matter and nutrients in different groups of cattle. The concentration of ammonia nitrogen (NH3-N) and short chain fatty acids (SCFA) in the rumen were higher and lower, respectively, for the group of buffaloes in relation to other experimental groups evaluated. When considering the excretion of total purine derivatives, buffaloes showed the lowest value compared to other genetic groups evaluated; about 61.76% of the total genetic group Nellore and 57.62% of the total genetic group Holstein with an average of 33.67 mmol/day. For the buffaloes, the excretion of xanthine and hypoxanthine observed was of 5.11% of total purine derivatives. There was a better nitrogen balance (g/day) for groups of Holstein heifers and Nellore in relation to the group of buffalo heifers. There were differences in the concentrations of urea and urea nitrogen in serum and liver enzymes where the buffaloes had higher values in relation at the bovines. There is a great metabolic diversity among the experimental groups evaluated and it was more exacerbated among buffaloes and bovines, when submitted to the same diet and same management conditions. (C) 2011 Elsevier B.V. All rights reserved.

The IWA Anaerobic Digestion Model No 1 (ADM1)

The IWA Anaerobic Digestion Modelling Task Group was established in 1997 at the 8th World Congress on,Anaerobic Digestion (Sendai, Japan) with the goal of developing a generalised anaerobic digestion model. The structured model includes multiple steps describing biochemical as well as physicochemical processes. The biochemical steps include disintegration from homogeneous particulates to carbohydrates, proteins and lipids; extracellular hydrolysis of these particulate substrates to sugars, amino acids, and long chain fatty acids (LCFA), respectively; acidogenesis from sugars and amino acids to volatile fatty acids (VFAs) and hydrogen; acetogenesis of LCFA and VFAs to acetate; and separate methanogenesis steps from acetate and hydrogen/CO2. The physico-chemical equations describe ion association and dissociation, and gas-liquid transfer. Implemented as a differential and algebraic equation (DAE) set, there are 26 dynamic state concentration variables, and 8 implicit algebraic variables per reactor vessel or element. Implemented as differential equations (DE) only, there are 32 dynamic concentration state variables.

Multi-elemental analysis of ready-to-eat “baby leaf” vegetables using microwave digestion and high-resolution continuum source atomic absorption spectrometry

The mineral content (phosphorous (P), potassium (K), sodium (Na), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn) and copper (Cu)) of eight ready-to-eat baby leaf vegetables was determined. The samples were subjected to microwave-assisted digestion and the minerals were quantified by High-Resolution Continuum Source Atomic Absorption Spectrometry (HR-CS-AAS) with flame and electrothermal atomisation. The methods were optimised and validated producing low LOQs, good repeatability and linearity, and recoveries, ranging from 91% to 110% for the minerals analysed. Phosphorous was determined by a standard colorimetric method. The accuracy of the method was checked by analysing a certified reference material; results were in agreement with the quantified value. The samples had a high content of potassium and calcium, but the principal mineral was iron. The mineral content was stable during storage and baby leaf vegetables could represent a good source of minerals in a balanced diet. A linear discriminant analysis was performed to compare the mineral profile obtained and showed, as expected, that the mineral content was similar between samples from the same family. The Linear Discriminant Analysis was able to discriminate different samples based on their mineral profile.

Effects of storage, processing and proteolytic digestion on microcystin-LR concentration in edible clams

Accumulation of microcystin-LR (MC-LR) in edible aquatic organisms, particularly in bivalves, is widely documented. In this study, the effects of food storage and processing conditions on the free MC-LR concentration in clams (Corbicula fluminea) fed MC-LR-producing Microcystisaeruginosa (1 × 105 cell/mL) for four days, and the bioaccessibility of MC-LR after in vitro proteolytic digestion were investigated. The concentration of free MC-LR in clams decreased sequentially over the time with unrefrigerated and refrigerated storage and increased with freezing storage. Overall, cooking for short periods of time resulted in a significantly higher concentration (P < 0.05) of free MC-LR in clams, specifically microwave (MW) radiation treatment for 0.5 (57.5%) and 1 min (59%) and boiling treatment for 5 (163.4%) and 15 min (213.4%). The bioaccessibility of MC-LR after proteolytic digestion was reduced to 83%, potentially because of MC-LR degradation by pancreatic enzymes. Our results suggest that risk assessment based on direct comparison between MC-LR concentrations determined in raw food products and the tolerable daily intake (TDI) value set for the MC-LR might not be representative of true human exposure.

Separation of bioactive peptides by electrodialysis with ultrafiltration membranes: membrane characteristics, ex-situ and in-situ digestion and their impact on peptide migration

Dissertation presented to Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa for obtaining the master degree in Membrane Engineering

Acidogenic digestion of effluents of the cheese industry in packed bed biofilm reactors

Dissertation for the Master degree in Biotechnology

In vitro digestion and stability assessment of b-lactoglobulin/riboflavin nanostructures

B-Lactoglobulin (b-Lg) is the major protein fraction of bovine whey serum and a primary gelling agent. b-Lg has a high nutritional value, is stable at low pH being highly resistant to proteolytic degradation in the stomach, besides, it has the ability of acting as an encapsulating agent. This study aims at assessing the ability of b-Lg nanostructures to associate a nutraceutical - i.e. riboflavin - and release it in a controlled manner throughout an in vitro gastrointestinal (GI) system. For this reason b-Lg nanostructures loaded with riboflavin were critically characterized in terms of their morphology (i.e. size, polydispersity, -potential and shape) by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and efficiency to associate to riboflavin through spectrofluorimetry. Furthermore, these nanocomplexes were evaluated in an in vitro GI model, simulating the physiological conditions. Stable b-Lg nanostructures were obtained at pH 6, of spherical shape, characterized by particle size of 172±1 nm, low polydispersity (i.e. PDI of 0.06±0.02), -potential of -32±3 mV and association efficiency (AE) of 26±1 %. b-Lg nanostructures showed to be stable upon their passage throughout stomach (i.e. particle size, PDI and potential of 248±10 nm, 0.18±0.03 and 18±3 mV, respectively). Concerning their passage throughout the intestine, such nanostructures were mostly degraded in the duodenum. Regarding riboflavin, a release of about 11 % was observed after their passage through stomach, while 35 %, 38 % and 5 % were the released percentages of the total riboflavin associated observed after passage through duodenum, jejunum and ileum, respectively. Hence,b-Lg nanostructures showed to be suitable carriers for riboflavin until the intestine, where their degradation occurs. b-Lg also showed to be structurally stable, under food simulant conditions (yoghurt simulant, composed of 3 % acetic acid), over 14 days, with a protective effect upon riboflavin activity, releasing it in a 7 day period.

The appraisal of mesophilic anaerobic digestion and thermophilic aerobic digestion in the treatment of municipal sludge in Ireland

It has been well documented that the optimum feedstock for anaerobic digesters consists of readily biodegradable compounds, as found in primary sludge or even a mixed substrate of primary and excess activated sludge. Due to the requirements of the Urban Wastewater Treatment Plant Directive of 1991, the quantities of secondary sludge generated is set to increase substantially. A pilot scale study was undertaken to evaluate the performance of both Mesophilic Anaerobic Digestion and Thermophilic Aerobic digestion in the treatment of secondary sludge. The results indicated that the anaerobic pilot scale digester achieved a greater solids destruction than the aerobic pilot plant averaging at 28% T.S. removal verses 20% for the aerobic digester, despite the fact that secondary sludge is the optimum feedstock for aerobic digestion. This can, however, be attributed to the greater biomass yield experienced with aerobic systems, and to the absence of Autothermal conditions. At present, the traditional technique of Mesophilic Anaerobic Digestion is in widespread application throughout Ireland, for the stabilisation of sewage sludge. There is only one Autothermal Thermophilic Aerobic Digester at present situated in Killarney, Co. Kerry. A further objectives of the study was to compare full-scale applications of Mesophilic Anaerobic Digestion to ATAD. Two Sludge Treatment plants, situated in Co. Kerry, were used for this purpose, and were assessed mainly under the following headings; process stability, solids reduction on average, the ATAD plant in Killarney has the advantage of producing a “Class A” Biosolid in terms of pathogen reduction, and can effectively treat double the quantity of sludge. In addition, economically the ATAD plant is cheaper to run, costing €190 / t.d.s verses €211 / t.d.s. for the anaerobic digester in Tralee. An overview of additional operational Anaerobic Digestion Plants throughout Ireland is also presented.

The appraisal of anaerobic digestion in Ireland to develop improved designs and operational practice

Mesophilic Anaerobic Digestion treating sewage sludge was investigated at five full-scale sewage treatment plants in Ireland. The anaerobic digestion plants are compared and evaluated in terms of design, equipment, operation, monitoring and management. All digesters are cylindrical, gas mixed and heated Continuously Stirred Tank Reactors (CSTR), varying in size from 130m3 to 800m3. Heat exchanger systems heat all digesters. Three plants reported difficulties with the heating systems ranging from blockages to insufficient insulation and design. Exchangers were modified and replaced within one year of operation at two plants. All but one plant had Combined Heat and Power (CHP) systems installed. Parameter monitoring is a problem at all plants mainly due to a lack of staff and knowledge. The plant operators consider pH and temperature the most important parameters to be measured in terms of successful monitoring of an anaerobic digester. The short time taken and the ease at which pH and temperature can be measured may favour these parameters. Three laboratory scale pilot anaerobic digesters were operated using a variety of feeds over at 144-day period. Two of the pilots were unmixed and the third was mechanically mixed. As expected the unmixed reactors removed more COD by retention of solids in the digesters but also produced greater quantities of biogas than the mixed digester, especially when low solids feed such as whey was used. The mixed digester broke down more solids due to the superior contact between the substrate and the biomass. All three reactors showed good performance results for whey and sewage solids. Scum formation occurred giving operational problems for mixed and unmixed reactors when cattle slurry was used as the main feed source. The pilot test was also used to investigate which parameters were the best indicators of process instability. These trials clearly indicated that total Volatile Fatty Acid (VFA) concentrations was the best parameter to show signs of early process imbalance, while methane composition in the biogas was good to indicate possible nutrient deficiencies in the feed and oxygen shocks. pH was found to be a good process parameter only if the wastewater being treated produced low bicarbonate alkalinities during treatment.

Comparison between Two Methods for Diagnosis of Trichinellosis: Trichinoscopy and Artificial Digestion

Two direct methods for the diagnosis of trichinellosis were compared: trichinoscopy and artificial digestion. Muscles from 17 wistar rats, orally infected with 500 Trichinella spiralis encysted larvae were examined. From each of the following muscles: diaphragm, tongue, masseters, intercostals, triceps brachialis and cuadriceps femoralis, 648,440 larvae from 1 g samples were recovered. The linear correlation between trichinoscopy and artificial digestion was very high and significant (r=0.94, p< 0.0001), showing that both methods for the detection of muscular larvae did not differ significantly. In both methods, significant differences were found in the distribution of larvae per gramme of muscle