Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.     

Resuscitation of Escherichia coli VBNC cells depends on a variety of environmental or chemical stimuli

Aims: To analyse viable but nonculturable (VBNC) state induction in Escherichia coli and resuscitation of VBNC suspensions in several conditions. Methods and Results: VBNC were induced in four media, two temperatures and six strains, but only cells produced at 4°C were able to resuscitate. Resuscitation of 14 VBNC suspensions obtained in several conditions occurred in the presence of supernatants of growing cells, in minimal medium supplemented with amino acids or after temperature change, depending on strain. A limited time period beyond no more resuscitation that could be observed was also confirmed. Conclusions: The supernatants positive effect is suggested to be because of a nonproteinaceous molecule, and a combination of methionine, glutamine, threonine, serine and asparagine could be used as primary mix for resuscitation experiments. Significance and Impact of the Study: Escherichia coli resuscitation was already attempted in several conditions, but it is the first time that a positive result was observed in minimal medium supplemented with amino acids or after temperature change. The role of amino acids in resuscitation is of special interest since was never reported for any species.     

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

Aims: A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. Methods and Results: A total of 30 clones exhibiting activity on α‐naphthyl acetate (α‐NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase‐coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro‐organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p‐nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Conclusions: Fae1A exhibited a lower K_m and higher catalytic efficiency (k_cat/K_m) on ferulic acid esters than on α‐NA or p‐nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p‐coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine‐specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. Significance and Impact of the Study: To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.     

Incidence and survival of non-O157 verocytotoxigenic Escherichia coli in soil

Aims: This study estimated the incidence of non‐O157 verocytotoxigenic Escherichia coli (VTEC) in farm pasture soils and investigated the survival of non‐O157 VTEC in clay and sandy loam soils. Methods and Results: Twenty farms were tested over a 12‐month period by sample enrichment in tryptone soya broth plus vancomycin, followed by PCR screening for the presence of vt1 and vt2 genes. Of the 600 soil samples, 162 (27%), across all farms, were found to contain vt1 and/or vt2 genes. The enrichment cultures from the 162 PCR‐positive samples were plated onto Chromocult tryptone bile X‐glucuronide agar (TBX), presumptive VTEC colonies recovered, confirmed as VTEC by PCR and serotyped. Samples of the two predominant soil types in Ireland (clay and sandy) were homogenized, characterized in terms of pH, boron, cobalt, copper, potassium, magnesium, manganese, phosphorus, zinc and organic matter content, inoculated with washed suspensions of eight non‐O157:H7 soil isolates and six bovine faecal isolates and stored at 10°C for up to 201 days. Inoculum survival rates were determined at regular intervals by recovering and plating soil samples on TBX. All inoculated non‐O157 serotypes had highest D‐values in the sandy loam soil with D‐values ranging from 50·26 to 75·60 days. The corresponding range in clay loam soils was 31·60–48·25 days. Conclusions: This study shows that non‐O157 VTEC occur widely and frequently in pasture soils and can persist in such environments for several months, with considerable opportunity for recycling through farm environments, and cattle, with clear potential for subsequent transmission into the human food chain. Significance and Impact of the Study: This is the first such study of non‐O157 VTEC in farm soils and found that these VTEC are frequent and persistent contaminants in farm soils. In light of recent epidemiological data, non‐O157 VTEC should be seen as an emerging risk to be controlled within the food chain.     

Identification and siting of native vegetation types on disturbed land: demonstration of statistical methods

Question: How does one best choose native vegetation types and site them in reclamation of disturbed sites ranging from cropland and strip mines? Application: World‐wide, demonstrated in SE Montana. Methods: We assumed that pre‐disturbance native communities are the best targets for revegetation, and that the environmental facet each occupies naturally provides its optimal habitat. Given this assumption, we used pre‐strip‐mine data (800 points from a 88 km² site) to demonstrate statistical methods for identifying native communities, describing them, and determining their environments. Results and conclusions: Classification and pruning analysis provided an objective method for choosing the number of target community types to be used in reclamation. The composition of eight target types, identified with these analyses, was described with a relevé table to provide a species list, target cover levels and support the choice of species to be seeded. As a basis for siting communities, we modeled community presence as a function of topography, slope/aspect, and substrate. Logistic GLMs identified the optimal environment for each community. Classification and Regression Tree (CART) analysis identified the most probable community in each environmental facet. Topography and slope were generally the best predictors in these models. Because our analyses relate native vegetation to undisturbed environments, our results may apply best to sites with minimal substrate disturbance (i.e. better to abandoned cropland than to strip‐mined sites).     

Global patterns of leaf mechanical properties

Leaf mechanical properties strongly influence leaf lifespan, plant-herbivore interactions, litter decomposition and nutrient cycling, but global patterns in their interspecific variation and underlying mechanisms remain poorly understood. We synthesize data across the three major measurement methods, permitting the first global analyses of leaf mechanics and associated traits, for 2819 species from 90 sites worldwide. Key measures of leaf mechanical resistance varied c. 500-800-fold among species. Contrary to a long-standing hypothesis, tropical leaves were not mechanically more resistant than temperate leaves. Leaf mechanical resistance was modestly related to rainfall and local light environment. By partitioning leaf mechanical resistance into three different components we discovered that toughness per density contributed a surprisingly large fraction to variation in mechanical resistance, larger than the fractions contributed by lamina thickness and tissue density. Higher toughness per density was associated with long leaf lifespan especially in forest understory. Seldom appreciated in the past, toughness per density is a key factor in leaf mechanical resistance, which itself influences plant-animal interactions and ecosystem functions across the globe.     

A fibre optic catheter for simultaneous measurement of longitudinal and circumferential muscular activity in the gastrointestinal tract

Diagnostic catheters based on fibre Bragg gratings (FBG's) are proving to be highly effective for measurement of the muscular activity associated with motility in the human gut. While the primary muscular contractions that generate peristalsis are circumferential in nature, it has long been known that there is also a component of longitudinal contractility present, acting in harmony with the circumferential component to improve the overall efficiency of material movement. We report the detection of longitudinal motion in mammalian intestine using an FBG technique that should be viable for similar detection in humans. The longitudinal sensors have been combined with our previously reported FBG pressure sensing elements to form a composite catheter that allows the relative phase between the two components to be detected. The catheter output has been validated using video mapping in an ex‐vivo rabbit ileum preparation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Patterns of tree phenological diversity in dry tropics

Recent phenological studies in tropical deciduous forests revealed a mosaic of vegetation composed of several pheno-phases that are evolved as an adaptation by the species to overcome seasonal drought in different ways. These pheno-phases represent extent of annual deciduousness (～leaflessness) and triggering factors for buds break (e.g. vegetative and flower). Thus, studying patterns of various pheno-phases (phonological diversity) in tropical forest have been thought to provide a potential tool to address critical questions related to climate change modeling and monitoring. In tropics, tree species represent a gradient of deciduousness (from leaf-exchanging species to >6 months deciduous species) and flowering initiation (breaking of flower buds in various part of annual cycle). Both processes are mostly triggered by variation in day length and/or temperature during late dry season/autumn, and/or first significant rain during rainy season. In addition, few factors like drought induced leaf fall and sporadic winter rains are supposed to affect these processes temporarily. Besides, the abundances of pheno-phases (i.e. leafing and flowering) also vary among tropical deciduous forest trees. Presence of such variations in tropical tree pheno-phases and their abundances are reported to vary due to micro-climatic variables and has specific implications in tropical forests. Present paper discusses the existing information on various pheno-phases and their abundances in tropical forests and role of climatic factors on tree phonological diversity. Further, we emphasized the need to develop predicting understanding of impending climatic change (i.e. precipitation and temperature) on diversity of pheno-phases by collecting long-term data on tree pheno-phases through a network of phonological stations in dry tropics.     

Effect of decreasing dietary protein level and replacing starch with soluble fibre on digestive physiology and performance of growing rabbits

At weaning (33 days of age), 246 hybrid rabbits (782 ± 53 g live weight) were divided into six experimental groups and fed ad libitum six iso-ADF diets formulated according to a bifactorial arrangement with two protein levels (152 and 162 g/kg) and three soluble fibre-to-starch ratios (0.2, 0.6 and 1.5), the latter obtained by replacing starch (from 209 to 91 g/kg) with soluble fibre (from 48 to 136 g/kg). The trial lasted for 42 days until slaughter. The rabbits that were fed the diet with the highest protein level and the lowest soluble fibre-to-starch ratio showed the highest mortality rate (17.1% v. 1.7% on average; P < 0.001) and sanitary risk (mortality + morbidity: 20.0% v. 8.1%; P = 0.04) compared with the rabbits fed the other diets. With increasing dietary crude protein level, the digestibility of dry matter (DM; 0.615 to 0.626) and gross energy (0.620 to 0.630) as well as aNDF (without sodium sulphite; 0.298 to 0.323) and hemicelluloses (0.417 to 0.461) significantly (0.001 < P < 0.10) improved. Moreover, total volatile fatty acids (VFAs) in the caecal content increased (59.0 to 68.4 mmol/l; P = 0.01) and ileum crypt depth tended to reduce (P = 0.07). Neither growth performance nor slaughter results were affected by the protein level. When increasing soluble fibre-to-starch ratio, the digestibility of DM and gross energy did not change, whereas the digestibility of aNDF (0.264 to 0.352), ADF (0.167 to 0.267) and hemicelluloses (0.400 to 0.470) linearly increased (P < 0.001). At caecum, N-ammonia tended to decrease linearly (P = 0.08), total VFA concentration (56.0 to 67.3 mmol/l) and acetate proportion (80.4 to 83.3 mmol/100 mmol VFA) linearly increased (P < 0.01), whereas butyrate and valerate proportions decreased (0.01 < P < 0.05). Growth performance was similar among groups, whereas at slaughter the proportion of the gastrointestinal tract linearly increased (177 to 184 g/kg; P < 0.01) without effect on dressing percentage, however. As soluble fibre-to-starch ratio increased, meat pH linearly decreased and lightness (L*), redness (a*) and yellowness (b*) colour indexes increased (0.01 < P < 0.05).     

Variation in meat quality characteristics between Sanga (Bos taurus africanus) and Sanga-derived cattle breeds and between Sanga and Brahman (Bos indicus)

Cattle breeds indigenous to Africa (Sanga) compare favourably to Bos indicus breeds with regard to adaptation to harsh environments. This study compared the meat quality of three Sanga breeds (Nguni, Tuli and Drakensberger), a Sanga-related breed (Bonsmara) and a B. indicus breed (Brahman) and supported these results with biochemical and histological measurements on the M. longissimus lumborum. Twelve young grain-fed steers of each breed were slaughtered and carcasses were electrically stimulated. All Sanga (and related) breeds, with the exception of the Tuli, had lower Warner-Bratzler shear force (SF) values at 2 and 21 days post mortem compared with the BR (P < 0.05). Measurements related to the calcium-dependent protease system and myofibrillar fragmentation explained the bulk of the variation among breeds, whereas variation in fibre type, sarcomere length and connective tissue properties gave less convincing support. With the exception of the Tuli, Sanga and Sanga-related breeds produced more tender (according to SF) meat than BR, mainly due to favourable calpain-to-calpastatin ratios. Small differences in colour, drip loss and cooking properties were found among breeds (P < 0.05).