Didactics–promoting collaborative work and personal learning environment. Critical, creative and autonomous roles in a competitive society

School has evolved from a place where knowledge is provided to a place where learners are helped to develop their professional and social skills. Consequently, education must evolve through big challenges in order to face the changes of society in the XXIst century

O potencial da PCR em tempo real no diagnóstico da infecção por helicobacter pylori

H. pylori é um microrganismo responsável por gastrites e implicado, em associação com outros factores, na úlcera gastroduodenal e no cancro gástrico. O diagnóstico da infecção por microrganismo pode realizar-se recorrendo a métodos invasivos através da obtenção de uma biópsia gástrica obtida por endoscopia digestiva alta e a métodos não invasivos. Nenhum dos métodos, desenvolvido até hoje, constitui o método ideal. Todos eles possuem as suas vantagens e desvantagens consoante a situação em que são aplicados. A reacção de polimerização em cadeia (PCR) conduziu a uma modificação fundamental no campo da biologia molecular, abrindo novos horizontes nas ciências médicas e biológicas. Apesar da cultura de H. pylori a partir de biópsia gástrica continuar a ser o método de referência para o diagnóstico da infecção por esta bactéria, ela apresenta inconvenientes que podem ser ultrapassados pela utilização da PCR, como sejam o longo período para a obtenção de resultados e o respeito de condições estritas de transporte da biópsia gástrica. Recentemente foi desenvolvido um protocolo baseado no principio da PCR em tempo real, utilizando o aparelho LightCycler Roche Diagnostics. Este protocolo permite a obtenção de um resultado de detecção da presença de H. pylori na biópsia gástrica assim como do seu perfil de susceptibilidade aos macrólidos. A PCR em tempo real é dotada de uma grande sensibilidade e especificidade, rapidez de obtenção de resultados o que aliado à sua capacidade de detecção de mutações responsáveis pela resistência dos microrganismos aos antibióticos faz com que esta técnica seja a metodologia do futuro no diagnóstico das doenças infecciosas.

The biocontrol fungus Pochonia chlamydosporia shows nematode host preference at the infraspecific level

A RAPD-PCR assay was developed and used to test For competitive variability in growth of the nematode biological control fungus Pochonia chlamydosporia. Saprophytic competence in soil with or without tomato plants was examined in three isolates of the fungus: RES 280 (J), originally isolated from potato cyst nematode (PCN) cysts; RES 200 (1) and RES 279 (S), both originally isolated from root knot nematode (RKN) eggs. Viable counts taken at 70 d indicated that I was the best saprophyte followed by S, with J the poorest. RAPD-PCR analysis of colonies from mixed treatments revealed that there was a cumulative effect of adding isolates to the system. This Suggested that the isolates did not interact and that they may occupy separate niches in soil and the rhizosphere. To investigate parasitic ability, soils were seeded with two isolates of the fungus: J and S, singly or in combination. Tomato or potato plants were grown in these soils; free of nematodes, or inoculated with PCN or RKN, and incubated for 77 d. The abundance of the PCN isolate J in PCN cysts was significantly greater than that of the RKN isolate S but in RKN egg masses, S was significantly more abundant than J. RAPD-PCR analysis of colonies from mixed treatments confirmed that J was more abundant than S ill PCN cysts whereas the converse was observed on RKN egg masses. This substantiates the phenomenon of nematode host preference at the infraspecific level of P. chlamydosporia and highlights its relevance for biological control of plant parasitic nematodes.

Evaluation of PCR to detect Theileria parva in field-collected tick and bovine samples in Tanzania

The ability of PCR to detect infections of Theileria parva, the cause of East Coast Fever, in field-collected tick and bovine samples from Tanzania was evaluated. PCR-detected infection prevalence was high (15/20, 75%) in unfed adult Rhipicephalus appendiculatus ticks that fed as nymphs on an acutely-infected calf, but low (22/836, 2.6%) in unfed adult R. appendiculatus collected from field sites in Tanzania. Tick infection prevalence was comparable to that in previous studies that used salivary gland staining to detect T parva infection in field-collected host-seeking ticks. Of 282 naturally-exposed zebu calves, seven had PCR-positive buffy coat samples prior to detection of Theileria spp. parasites in stained huffy coat cells or lymph node biopsies. Evidence of Theileria spp. infections was detected in stained smears of lymph node biopsies from 109 calves (38.6%) and huffy coat samples from 81 (28.7%), while huffy coat samples from 66 (23.4%) were PCR-positive for T parva. Implications of these findings for the sensitivity and specificity of the PCR are discussed. (C) 2003 Elsevier Science B.V. All rights reserved.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

The influence of winter oilseed rape (Brassica napus ssp oleifera var. biennis) cultivar and grass genotype on the competitive balance between crop and grass weeds

Three experiments conducted over two years (2002-04) at the Crops Research Unit, University of Reading, investigated competition between autumn sown oilseed rape cultivars (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) and Lolium multiflorum Lam., L. x boucheanum Kunth and Alopecurus myosuroides Huds., sown as indicative grass weeds. Rape cultivar (cv.) had a substantial effect on grass weed seed return. Over the six cultivars tested, L. multiflorum spikelet production ranged from just under 400 spikelets/m(2) in the presence of cv. Winner to nearly 5800 in competition with cv. Lutin. Cultivar competitiveness was associated with high biomass, large dense floral layers and early stem extension. There was some evidence of differential competitive tolerance between rape cultivars. The results suggested that rape cultivars could be screened for competitiveness by measuring floral layer interception of photosynthetic active radiation. L. x boucheanum cultivars varied in ability to compete with rape. In the absence of inter-specific competition, spikelet density was similar for Aberecho and Polly (circa 31000 spikelets/m(2)) but when grown with rape Polly outyielded Aberecho (i.e. 12 090 and 7990 spikelets/m(2) respectively).

The potential for competitive and healthy food chains of benefit to the countryside

Agricultural policy liberalisation, concern about unhealthy diets and growing recognition of the importance of sustainable land use have fostered interest in the development of competitive food chains based around products that are beneficial to the rural environment. We review the potential for foods with enhanced health attributes based on alternative varieties/breeds and production systems to traditional agriculture which has been predominantly motivated by yields. We concentrate on soft fruit, which is an important source of polyphenols, and grazing livestock systems that have the potential for improving fatty acid profiles in meat products and find there to be clear scientific potential, but limited research to date. Consumer research suggests considerable acceptance of such products and willingness to pay sufficient to cover additional production costs. Purchase of such foods could have major implications for agricultural land use and the rural environment. There is little research to date on specific healthier food products, but spatially explicit models are being developed to assess land use and environmental implications of changing demand and husbandry methods.

Molecule modeling of human pentameric alpha(7) neuronal nicotinic acetylcholine receptor and its interaction with its agonist and competitive antagonist

The nicotinic Acetylcholine Receptor (nAChR) is the major class of neurotransmitter receptors that is involved in many neurodegenerative conditions such as schizophrenia, Alzheimer's and Parkinson's diseases. The N-terminal region or Ligand Binding Domain (LBD) of nAChR is located at pre- and post-synaptic nervous system, which mediates synaptic transmission. nAChR acts as the drug target for agonist and competitive antagonist molecules that modulate signal transmission at the nerve terminals. Based on Acetylcholine Binding Protein (AChBP) from Lymnea stagnalis as the structural template, the homology modeling approach was carried out to build three dimensional model of the N-terminal region of human alpha(7)nAChR. This theoretical model is an assembly of five alpha(7) subunits with 5 fold axis symmetry, constituting a channel, with the binding picket present at the interface region of the subunits. alpha-netlrotoxin is a potent nAChR competitive antagonist that readily blocks the channel resulting in paralysis. The molecular interaction of alpha-Bungarotoxin, a long chain alpha-neurotoxin from (Bungarus multicinctus) and human alpha(7)nAChR seas studied. Agonists such as acetylcholine, nicotine, which are used in it diverse array of biological activities, such as enhancements of cognitive performances, were also docked with the theoretical model of human alpha(7)nAChR. These docked complexes were analyzed further for identifying the crucial residues involved in interaction. These results provide the details of interaction of agonists and competitive antagonists with three dimensional model of the N-terminal region of human alpha(7)nAChR and thereby point to the design of novel lead compounds.

Application of real-time and multiplex PCR assays to study leaf blotch epidemics in barley

Leaf blotch, caused by Rhynchosporium secalis, was studied in a range of winter barley cultivars using a combination of traditional plant pathological techniques and newly developed multiplex and real-time polymerase chain reaction (PCR) assays. Using PCR, symptomless leaf blotch colonization was shown to occur throughout the growing season in the resistant winter barley cv. Leonie. The dynamics of colonization throughout the growing season were similar in both Leonie and Vertige, a susceptible cultivar. However, pathogen DNA levels were approximately 10-fold higher in the susceptible cultivar, which expressed symptoms throughout the growing season. Visual assessments and PCR also were used to determine levels of R. secalis colonization and infection in samples from a field experiment used to test a range of winter barley cultivars with different levels of leaf blotch resistance. The correlation between the PCR and visual assessment data was better at higher infection levels (R(2) = 0.81 for leaf samples with >0.3% disease). Although resistance ratings did not correlate well with levels of disease for all cultivars tested, low levels of infection were observed in the cultivar with the highest resistance rating and high levels of infection in the cultivar with the lowest resistance rating.

The influence of winter oilseed rape (Brassica napus ssp. oleifera var. biennis) cultivar and grass genotype on the competitive balance between crop and grass weeds

Four experiments conducted over three seasons (2002–05) at the Crops Research Unit, University of Reading, investigated effects of canopy management of autumn sown oilseed rape (Brassica napus L. ssp. oleifera var. biennis (DC.) Metzg.) on competition with grass weeds. Emphasis was placed on the effect of the crop on the weeds. Rape canopy size was manipulated using sowing date, seed rate and the application of autumn fertilizer. Lolium multiflorum Lam., L.rboucheanum Kunth and Alopecurus myosuroides Huds. were sown as indicative grass weeds. The effects of sowing date, seed rate and autumn nitrogen on crop competitive ability were correlated with rape biomass and fractional interception of photosynthetically active radiation (PAR) by the rape floral layer, to the extent that by spring there was good evidence of crop: weed replacement. An increase in seed rate up to the highest plant densities tested increased both rape biomass and competitiveness, e.g. in 2002/3, L. multiflorum head density was reduced from 539 to 245 heads/m2 and spikelet density from 13 170 to 5960 spikelets/m2 when rape plant density was increased from 16 to 81 plants/m2. Spikelets/head of Lolium spp. was little affected by rape seed rate, but the length of heads of A. myosuroides was reduced by 9%when plant density was increased from 29–51 plants/m2. Autumn nitrogen increased rape biomass and reduced L. multiflorum head density (415 and 336 heads/m2 without and with autumn nitrogen, respectively) and spikelet density (9990 and 8220 spikelets/m2 without and with autumn nitrogen, respectively). The number of spikelets/head was not significantly affected by autumn nitrogen. Early sowing could increase biomass and competitiveness, but poor crop establishment sometimes overrode the effect. Where crop and weed establishment was similar for both sowing dates, a 2-week delay (i.e. early September to mid-September) increased L. multiflorum head density from 226 to 633 heads/m2 and spikelet density from 5780 to 15 060 spikelets/m2.