In vivo expression technology (IVET) selection of genes of Rhizobium leguminosarum biovar viciae A34 expressed in the rhizosphere

IVET was used to identify genes that are specifically expressed in the rhizosphere of the pea-nodulating bacterium Rhizobium leguminosarum A34. A library of R. leguminosarum A34 cloned in the integration vector pIE1, with inserts upstream of a promoter-less purN:gfp:gusA, was conjugated into purN host RU2249 and recombined into the genome. After removal of colonies that expressed the reporter genes of the vector under laboratory conditions, the library was inoculated into a nonsterile pea rhizosphere. The key result is that 29 rhizosphere-induced loci were identified. Sequence analysis of these clones showed that a wide variety of R. leguminosarum A34 genes are expressed specifically in the rhizosphere including those encoding proteins involved in environmental sensing, control of gene expression, metabolic reactions and membrane transport. These genes are likely to be important for survival and colonization of the pea rhizosphere.

Characterization of the quaternary amine transporters of Rhizobium leguminosarum bv. viciae 3841

Rhizobium leguminosarum bv. viciae 3841 contains six putative quaternary ammonium transporters (Qat), of the ABC family. Qat6 was strongly induced by hyperosmosis although the solute transported was not identified. All six systems were induced by the quaternary amines choline and glycine betaine. It was confirmed by microarray analysis of the genome that pRL100079-83 (qat6) is the most strongly upregulated transport system under osmotic stress, although other transporters and 104 genes are more than threefold upregulated. A range of quaternary ammonium compounds were tested but all failed to improve growth of strain 3841 under hyperosmotic stress. One Qat system (gbcXWV) was induced 20-fold by glycine betaine and choline and a Tn5::gbcW mutant was severely impaired for both transport and growth on these compounds, demonstrating that it is the principal system for their use as carbon and nitrogen sources. It transports glycine betaine and choline with a high affinity (apparent K-m, 168 and 294 nM, respectively).

Thiamine is synthesized by a salvage pathway in Rhizobium leguminosarum bv. viciae strain 3841

In the absence of added thiamine, Rhizobium leguminosarum bv. viciae strain 3841 does not grow in liquid medium and forms only "pin" colonies on agar plates, which contrasts with the good growth of Sinorhizobium meliloti 1021, Mesorhizobium loti 303099, and Rhizobium etli CFN42. These last three organisms have thiCOGE genes, which are essential for de novo thiamine synthesis. While R. leguminosarum bv. viciae 3841 lacks thiCOGE, it does have thiMED. Mutation of thiM prevented formation of pin colonies on agar plates lacking added thiamine, suggesting thiamine intermediates are normally present. The putative functions of ThiM, ThiE, and ThiD are 4-methyl-5-(beta-hydroxyethyl) thiazole (THZ) kinase, thiamine phosphate pyrophosphorylase, and 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) kinase, respectively. This suggests that a salvage pathway operates in R. leguminosarum, and addition of HMP and THZ enabled growth at the same rate as that enabled by thiamine in strain 3841 but elicited no growth in the thiM mutant (RU2459). There is a putative thi box sequence immediately upstream of the thiM, and a gfp-mut3.1 fusion to it revealed the presence of a promoter that is strongly repressed by thiamine. Using fluorescent microscopy and quantitative reverse transcription-PCR, it was shown that thiM is expressed in the rhizosphere of vetch and pea plants, indicating limitation for thiamine. Pea plants infected by RU2459 were not impaired in nodulation or nitrogen fixation. However, colonization of the pea rhizosphere by the thiM mutant was impaired relative to that of the wild type. Overall, the results show that a thiamine salvage pathway operates to enable growth of Rhizobium leguminosarum in the rhizosphere, allowing its survival when thiamine is limiting.

Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids

Rhizobium leguminosarum synthesizes polyhydroxybutyrate and glycogen as its main carbon storage compounds. To examine the role of these compounds in bacteroid development and in symbiotic efficiency, single and double mutants of R. legumosarum bv. viciae were made which lack polyhydroxybutyrate synthase (phaC), glycogen synthase (glgA), or both. For comparison, a single phaC mutant also was isolated in a bean-nodulating strain of R. leguminosarum bv. phaseoli. In one large glasshouse trial, the growth of pea plants inoculated with the R. leguminosarum bv. viciae phaC mutant were significantly reduced compared with wild-type-inoculated plants. However, in subsequent glasshouse and growth-room studies, the growth of pea plants inoculated with the mutant were similar to wildtype-inoculated plants. Bean plants were unaffected by the loss of polyhydroxybutyrate biosynthesis in bacteroids. Pea plants nodulated by a glycogen synthase mutants or the glgA/phaC double mutant, grew as well as the wild type in growth-room experiments. Light and electron micrographs revealed that pea nodules infected with the glgA mutant accumulated large amounts of starch in the II/III interzone. This suggests that glycogen may be the dominant carbon storage compound in pea bacteroids. Polyhydroxybutyrate was present in bacteria in the infection thread of pea plants but was broken down during bacteroid formation. In nodules infected with a phaC mutant of R. leguminosarum bv. viciae, there was a drop in the amount of starch in the II/III interzone, where bacteroids form. Therefore, we propose a carbon burst hypothesis for bacteroid formation, where polyhydroxybutyrate accumulated by bacteria is degraded to fuel bacteroid differentiation.

Characterization of wheat puroindoline proteins

Puroindoline proteins were purified from selected UK-grown hexaploid wheats. Their identities were confirmed on the basis of capillary electrophoresis mobilities, relative molecular mass and N-terminal amino acid sequencing. Only one form of puroindoline-a protein was found in those varieties, regardless of endosperm texture. Three allelic forms of puroindoline-b protein were identified. Nucleotide sequencing of cDNA produced by RT-PCR of isolated mRNA indicated that these were the 'wild-type', found in soft wheats, puroindoline-b containing a Gly -> Ser amino acid substitution (position 46) and puroindoline-b containing a Trp -> Arg substitution (position 44). The latter two were found in hard wheats. Microheterogeneity, due to short extensions and/or truncations at the N-terminus and C-terminus, was detected for both puroindoline-a and puroindoline-b. The type of microheterogeneity observed was more consistent for puroindoline-a than for puroindoline-b, and may arise through slightly different post-translational processing pathways. A puroindoline-b allele corresponding to a Leu -> Pro substitution (position 60) was identified from the cDNA sequence of the hard variety Chablis, but no mature puroindoline-b protein was found in this or two other European varieties known to possess this puroindoline-b allele. Wheats possessing the puroindoline-b proteins with point mutations appeared to contain lower amounts of puroindoline protein. Such wheats have a hard endosperm texture, as do wheats from which puroindoline-a or puroindoline-b are absent. Our results suggest that point mutations in puroindoline-b genes may confer hard endosperm texture through accumulation of allelic forms of puroindoline-b proteins with altered functional properties and/or through lower amounts of puroindoline proteins.

Whole-grain wheat breakfast cereal has a prebiotic effect on the human gut microbiota: a double-blind, placebo-controlled, crossover study

Epidemiological studies have shown an inverse association between dietary intake of whole grains and the risk of chronic disease. This may be related to the ability to mediate a prebiotic modulation of gut microbiota. However, no studies have been conducted on the microbiota modulatory capability of whole-grain (WG) cereals. In the present study, the impact of WG wheat on the human intestinal microbiota compared to wheat bran (WB) was determined. A double-blind, randomised, crossover study was carried out in thirty-one volunteers who were randomised into two groups and consumed daily 48g breakfast cereals, either WG or WB, in two 3-week study periods, separated by a 2-week washout period. Numbers of faecal bifidobacteria and lactobacilli (the target genera for prebiotic intake), were significantly higher upon WG ingestion compared with WB. Ingestion of both breakfast cereals resulted in a significant increase in ferulic acid concentrations in blood but no discernible difference in faeces or urine. No significant differences in faecal SCFA, fasting blood glucose, insulin, total cholesterol (TC), TAG or HDL-cholesterol were observed upon ingestion of WG compared with WB. However, a significant reduction in TC was observed in volunteers in the top quartile of TC concentrations upon ingestion of either cereal. No adverse intestinal symptoms were reported and WB ingestion increased stool frequency. Daily consumption of WG wheat exerted a pronounced prebiotic effect on the human gut microbiota composition. This prebiotic activity may contribute towards the beneficial physiological effects of WG wheat.

Childhood emotional abuse and neglect as predictors of psychological and physical symptoms in women presenting to a primary care practice

Objective: There were two aims to this study: first to examine whether emotional abuse and neglect are significant predictors of psychological and somatic symptoms, and lifetime trauma exposure in women presenting to a primary care practice, and second to examine the strength of these relationships after controlling for the effects of other types of childhood abuse and trauma. Method: Two-hundred and five women completed the Childhood Trauma Questionnaire (Bernstein et al., 1994), Trauma History Questionnaire (Green, 1996), the Symptom Checklist-revised (Derogatis, 1997), and the Revised Civilian Mississippi Scale for posttraumatic stress disorder (Norris & Perilla, 1996) when presenting to their primary care physician for a visit. Hierarchical multiple regression analyses were conducted to examine unique contributions of emotional abuse and neglect variables on symptom measures while controlling for childhood sexual and physical abuse and lifetime trauma exposure. Results: A history of emotional abuse and neglect was associated with increased anxiety, depression, posttraumatic stress and physical symptoms, as well as lifetime trauma exposure. Physical and sexual abuse and lifetime trauma were also significant predictors of physical and psychological symptoms. Hierarchical multiple regressions demonstrated that emotional abuse and neglect predicted symptomatology in these women even when controlling for other types of abuse and lifetime trauma exposure. Conclusions: Long-standing behavioral consequences may arise as a result of childhood emotional abuse and neglect, specifically, poorer emotional and physical functioning, and vulnerability to further trauma exposure. (C) 2003 Elsevier Ltd. All rights reserved.

A classification system for delirium subtyping with the use of a commercial mobility monitor

The usefulness of motor subtypes of delirium is unclear due to inconsistency in subtyping methods and a lack of validation with objective measures of activity. The activity of 40 patients was measured over 24 h with a commercial accelerometer-based activity monitor. Accelerometry data from patients with DSM-IV delirium that were readily divided into hyperactive, hypoactive and mixed motor subtypes, were used to create classification trees that were Subsequently applied to the remaining cohort to define motoric subtypes. The classification trees used the periods of sitting/lying, standing, stepping and number of postural transitions as measured by the activity monitor as determining factors from which to classify the delirious cohort. The use of a classification system shows how delirium subtypes can be categorised in relation to overall activity and postural changes, which was one of the most discriminating measures examined. The classification system was also implemented to successfully define other patient motoric subtypes. Motor subtypes of delirium defined by observed ward behaviour differ in electronically measured activity levels. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

A continuous wavelet transform and classification method for delirium motoric subtyping

The usefulness of motor subtypes of delirium is unclear due to inconsistency in subtyping methods and a lack of validation with objective measures of activity. The activity of 40 patients was measured over 24 h with a discrete accelerometer-based activity monitor. The continuous wavelet transform (CWT) with various mother wavelets were applied to accelerometry data from three randomly selected patients with DSM-IV delirium that were readily divided into hyperactive, hypoactive, and mixed motor subtypes. A classification tree used the periods of overall movement as measured by the discrete accelerometer-based monitor as determining factors for which to classify these delirious patients. This data used to create the classification tree were based upon the minimum, maximum, standard deviation, and number of coefficient values, generated over a range of scales by the CWT. The classification tree was subsequently used to define the remaining motoric subtypes. The use of a classification system shows how delirium subtypes can be categorized in relation to overall motoric behavior. The classification system was also implemented to successfully define other patient motoric subtypes. Motor subtypes of delirium defined by observed ward behavior differ in electronically measured activity levels.

Validating a new clinical subtyping scheme for delirium with electronic motion analysis

The usefulness of motor subtypes of delirium is unclear due to inconsistency in sub-typing methods and a lack of validation with objective measures of activity. The activity of 40 patients was measured with 24 h accelerometry monitoring. Patients with Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) delirium (n = 30) were allocated into hyperactive, hypoactive and mixed motor subtypes. Delirium subtypes differed in relation to overall amount of activity, including movement in both sagittal and transverse planes. Differences were greater in the daytime and during the early evening ‘sundowning’ period. Frequency of postural changes was the most discriminating measure examined. Clinical subtypes of delirium defined by observed motor behaviour on the ward differ in electronically measured activity levels.

Experimental measurement and numerical simulation of horizontal-coupled slinky ground source heat exchangers

Results from both experimental measurements and 3D numerical simulations of Ground Source Heat Pump systems (GSHP) at a UK climate are presented. Experimental measurements of a horizontal-coupled slinky GSHP were undertaken in Talbot Cottage at Drayton St Leonard site, Oxfordshire, UK. The measured thermophysical properties of in situ soil were used in the CFD model. The thermal performance of slinky heat exchangers for the horizontal-coupled GSHP system for different coil diameters and slinky interval distances was investigated using a validated 3D model. Results from a two month period of monitoring the performance of the GSHP system showed that the COP decreased with the running time. The average COP of the horizontal-coupled GSHP was 2.5. The numerical prediction showed that there was no significant difference in the specific heat extraction of the slinky heat exchanger at different coil diameters. However, the larger the diameter of coil, the higher the heat extraction per meter length of soil. The specific heat extraction also increased, but the heat extraction per meter length of soil decreased with the increase of coil central interval distance.

Data based constructive identification - overcoming the curse of dimensionality

The modelling of a nonlinear stochastic dynamical processes from data involves solving the problems of data gathering, preprocessing, model architecture selection, learning or adaptation, parametric evaluation and model validation. For a given model architecture such as associative memory networks, a common problem in non-linear modelling is the problem of "the curse of dimensionality". A series of complementary data based constructive identification schemes, mainly based on but not limited to an operating point dependent fuzzy models, are introduced in this paper with the aim to overcome the curse of dimensionality. These include (i) a mixture of experts algorithm based on a forward constrained regression algorithm; (ii) an inherent parsimonious delaunay input space partition based piecewise local lineal modelling concept; (iii) a neurofuzzy model constructive approach based on forward orthogonal least squares and optimal experimental design and finally (iv) the neurofuzzy model construction algorithm based on basis functions that are Bézier Bernstein polynomial functions and the additive decomposition. Illustrative examples demonstrate their applicability, showing that the final major hurdle in data based modelling has almost been removed.

Determination of the in vivo prebiotic potential of a maize based wholegrain breakfast cereal: a human feeding study

Epidemiological studies have shown an inverse relationship between risk of CVD and intake of whole grain (WG)-rich food. Regular consumption of breakfast cereals can provide not only an increase in dietary WG but also improvements to cardiovascular health. Various mechanisms have been proposed, including prebiotic modulation of the colonic microbiota. In the present study, the prebiotic activity of a maize-derived WG cereal (WGM) was evaluated in a double-blind, placebo-controlled human feeding study (n 32). For a period of 21 d, healthy men and women, mean age 32 (sd 8) years and BMI 23·3 (sd 0·58) kg/m2, consumed either 48 g/d WG cereal (WGM) or 48 g placebo cereal (non-whole grain (NWG)) in a crossover fashion. Faecal samples were collected at five points during the study on days 0, 21, 42, 63 and 84 (representing at baseline, after both treatments and both wash-out periods). Faecal bacteriology was assessed using fluorescence in situ hybridisation with 16S rRNA oligonucleotide probes specific for Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum/perfringens subgroup, Lactobacillus–Enterococcus subgroup and total bacteria. After 21 d consumption of WGM, mean group levels of faecal bifidobacteria increased significantly compared with the control cereal (P = 0·001). After a 3-week wash-out period, bifidobacterial levels returned to pre-intervention levels. No statistically significant changes were observed in serum lipids, glucose or measures of faecal output. In conclusion, this WG maize-enriched breakfast cereal mediated a bifidogenic modulation of the gut microbiota, indicating a possible prebiotic mode of action