Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.)

<p>Abstract</p> <p>Background</p> <p>We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.</p> <p>Results</p> <p>A cDNA, named <it>aleh1</it>, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The <it>aleh1 </it>encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (<it>pI</it>) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of <it>aleh1 </it>was produced in <it>Pichia pastoris</it>, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.</p> <p>The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that <it>aleh1 </it>encoded 1-FEH.</p

Factors associated with physiotherapy provision in a population of elderly nursing home residents; a cross sectional study

BACKGROUND: Although physiotherapy (PT) plays an important role in improving activities of daily living (ADL functioning) and discharge rates, it is unclear how many nursing home residents receive treatment. Furthermore, there is a lack of insight into the determinants that influence the decision for treatment. In this study, we investigated how many nursing home residents receive PT. In addition, we analysed the factors that contribute to the variation in the provision of PT both between nursing homes and between residents. METHODS: A random sample of 600 elderly residents was taken from a random sample of 15 nursing homes. Residents had to be admitted for rehabilitation or for long-term care. Data were collected through interviews with the nursing home physician and the physiotherapist. Multilevel analysis was used to define the variation in the provision of PT and the factors that are associated with the question whether a resident receives PT or not. Furthermore the amount of PT provided was analysed and the factors that are associated with this. RESULTS: On average 69% of the residents received PT. The percentage of patients receiving treatment differed significantly across nursing homes, and especially the number of physiotherapists available, explained this difference between nursing homes. Residents admitted to a somatic ward for rehabilitation, and male residents in general, were most likely to receive PT. Residents who were treated by a physiotherapist received on average 55 minutes (sd 41) treatment a week. Residents admitted for rehabilitation received more PT a week, as were residents with a status after a total hip replacement. CONCLUSION: PT is most likely to be provided to residents on a somatic ward, recently admitted for rehabilitation to a nursing home, which has a relatively large number of physiotherapists. This suggests a potential under-use of PT for long-term residents with cognitive problems. It is recommended that physiotherapists reconsider which residents may benefit from treatment. This may require a shift in the focus of physiotherapists from 'recovery and discharge' to 'quality of life and well-being'

Linear viscoelastic behavior of aggregated colloidal dispersions

The viscoelastic behavior of a depletion-flocculated dispersion of colloidal spheres is investigated at different volume fractions of the spheres, using a controlled stress and a dynamic rheometer. Combining the results, we obtain the storage G′ and loss G′′ moduli over a frequency range of 0.02<ω<200rad/s. The measured G′ gradually increases with increasing frequency, while G′′ almost remains constant, indicating a broad spectrum of relaxation times. To describe and explain the observed behavior of the moduli as a function of frequency and volume fraction in terms of microscopic parameters, a microrheological model based on the fractal concept is proposed. Comparing experimental results with model calculations, we find a good agreement between the two, with physically plausible parameter values

Wheat genotypic variation in dynamic fluxes of WSC components in different stem segments under drought during grain filling

In wheat, stem water soluble carbohydrates (WSC), composed mainly of fructans, are the major carbon sources for grain filling during periods of decreasing photosynthesis or under drought stress after anthesis. Here, in a field drought experiment, WSC levels and associated enzyme activities were followed in different stem segments (peduncle, penultimate internode, lower parts of stem, and sheath) during grain filling. The focus was on two double haploid (DH) lines, DH 307 and DH 338, derived from a Westonia/Kauz cross, two drought-tolerant wheat varieties that follow different drought adaptation strategies during grain filling. The results showed that in irrigated plants, in the period between 20 and 30 days after anthesis (DAA), 70–80% of WSC were fructans. Before and after this period, the fructan proportion varied from 10 to 60%, depending on the location along the stem. Under drought, the fructan proportion changed, depending on genotype, and developmental stages. After anthesis, stem fructans accumulation occurred mainly in the peduncle and penultimate internode until 14 DAA in both DH lines, with clear genotypic variation in subsequent fructan degradation under drought. In DH 307 a significant reduction of fructans with a concomitant increase in fructose levels occurred earlier in the lower parts of the stem and the sheath, as compared to DH 338 or other stem segments in both lines. This was associated with an earlier increase of grain weight and thousand grain weight in DH 307. Spatiotemporal analysis of fructan dynamics and enzymatic activities in fructan metabolism revealed that several types of FEHs are involved in fructan remobilization to the grain under drought

Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)

The main storage compounds in Lolium perenne are fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare

Fecal Microbiota Transplantation from Overweight or Obese Donors in Cachectic Patients with Advanced Gastroesophageal Cancer : A Randomized, Double-blind, Placebo-Controlled, Phase II Study

Purpose Cachexia is a multifactorial syndrome, associated with poor survival in patients with cancer, and is influenced by the gut microbiota. We investigated the effects of fecal microbiota transplantation (FMT) on cachexia and treatment response in patients with advanced gastroesophageal cancer. Experimental Design: In a double-blind randomized placebo-controlled trial performed in the Amsterdam University Medical Center, we assigned 24 cachectic patients with metastatic HER2-negative gastroesophageal cancer to either allogenic FMT (healthy obese donor) or autologous FMT, prior to palliative chemotherapy (capecitabine and oxaliplatin). Primary objective was to assess the effect of allogenic FMT on satiety. Secondary outcomes were other features of cachexia, along with disease control rate (DCR), overall survival (OS), progression-free survival (PFS), and toxicity. Finally, exploratory analyses were performed on the effect of FMT on gut microbiota composition (metagenomic sequencing) and metabolites (untargeted metabolomics). Results: Allogenic FMT did not improve any of the cachexia outcomes. Patients in the allogenic group (n = 12) had a higher DCR at 12 weeks (P = 0.035) compared with the autologous group (n = 12), longer median OS of 365 versus 227 days [ HR = 0.38; 95% confidence interval (CI), 0.14-1.05; P = 0.057] and PFS of 204 versus 93 days (HR = 0.50; 95% CI, 0.21-1.20; P = 0.092). Patients in the allogenic group showed a significant shift in fecal microbiota composition after FMT (P = 0.010) indicating proper engraftment of the donor microbiota. Conclusions: FMT from a healthy obese donor prior to first-line chemotherapy did not affect cachexia, but may have improved response and survival in patients with metastatic gastroesophageal cancer. These results provide a rational for larger FMT trials.Peer reviewe

Are entrepreneurs' forecasts of economic indicators biased?

Insight into the investment behaviour of firms is central in understanding economic dynamics. A critical question, however, is whether firms provide sufficiently reliable data to enable them to make plausible forecasts at the meso (regional or sectoral) level. This paper analyses Dutch investment forecasts at different levels of aggregation. The central research question is whether entrepreneurs, individually or as a group, make systematic errors in their investment forecasts. A statistical test reveals that investment forecasts are not biased at the aggregated (regional and sectoral) level. At the micro level, however, there is a significant bias. Hence, using aggregated (regional and sectoral) data to test the lack of bias (unbiasedness) of forecasts may lead to the wrong conclusions. Moreover, aggregated investment forecasts may then be an inappropriate source for policy recommendations, despite their seemingly high reliability. This finding may in principle be valid for many European countries, since data collection on investment is organized in similar ways throughout Europe

Multi Locus Sequence Typing of Chlamydia Reveals an Association between Chlamydia psittaci Genotypes and Host Species

Chlamydia comprises a group of obligate intracellular bacterial parasites responsible for a variety of diseases in humans and animals, including several zoonoses. Chlamydia trachomatis causes diseases such as trachoma, urogenital infection and lymphogranuloma venereum with severe morbidity. Chlamydia pneumoniae is a common cause of community-acquired respiratory tract infections. Chlamydia psittaci, causing zoonotic pneumonia in humans, is usually hosted by birds, while Chlamydia abortus, causing abortion and fetal death in mammals, including humans, is mainly hosted by goats and sheep. We used multi-locus sequence typing to asses the population structure of Chlamydia. In total, 132 Chlamydia isolates were analyzed, including 60 C. trachomatis, 18 C. pneumoniae, 16 C. abortus, 34 C. psittaci and one of each of C. pecorum, C. caviae, C. muridarum and C. felis. Cluster analyses utilizing the Neighbour-Joining algorithm with the maximum composite likelihood model of concatenated sequences of 7 housekeeping fragments showed that C. psittaci 84/2334 isolated from a parrot grouped together with the C. abortus isolates from goats and sheep. Cluster analyses of the individual alleles showed that in all instances C. psittaci 84/2334 formed one group with C. abortus. Moving 84/2334 from the C. psittaci group to the C. abortus group resulted in a significant increase in the number of fixed differences and elimination of the number of shared mutations between C. psittaci and C. abortus. C. psittaci M56 from a muskrat branched separately from the main group of C. psittaci isolates. C. psittaci genotypes appeared to be associated with host species. The phylogentic tree of C. psittaci did not follow that of its host bird species, suggesting host species jumps. In conclusion, we report for the first time an association between C. psittaci genotypes with host species

A comparison of the measurement properties of the Juvenile Arthritis Functional Assessment Scale with the childhood health assessment questionnaire in daily practice

We compared the measurement properties of a performance test (Juvenile Arthritis Functional Assessment Scale; JAFAS) with a questionnaire-based instrument (Childhood Health Assessment Questionnaire; CHAQ) to measure functional ability in patients with juvenile idiopathic arthritis on the level of individual items. In 28 consecutive children visiting an outpatient paediatrics clinic, the JAFAS (range 0–20) and CHAQ (range 0–3) were applied, and measures of disease activity and joint range of motion (ROM) were determined. Twenty-eight children with a median age of 10 years and median disease duration of 3.2 years were included. The median JAFAS score was 0, and the median CHAQ score was 0.125. Cronbach’s alpha was 0.92 for the JAFAS and 0.96 for the CHAQ. The Spearman correlation coefficient between the JAFAS and the CHAQ was 0.55 (P < 0.01). With six out of ten items, the JAFAS classified the child as less disabled than with corresponding CHAQ activities. Overall, associations with measures of disease activity and ROM were higher for the CHAQ than for the JAFAS. A performance test (JAFAS) does not appear to have an added benefit over the questionnaire-based assessment (CHAQ) of physical function in a cross-sectional study