The level and distribution of methyl-esters influence the impact of pectin on intestinal T cells, microbiota, and Ahr activation.

Pectins are dietary fibres that modulate T cell immunity, microbiota composition, and fermentation profiles, but how this is influenced by the degree of methyl-esterification (DM) and degree-of-blockiness (DB) of pectin is unknown. Here, we demonstrate that supplementation of DM19(high-DB), DM49(low-DB) and DM43(high-DB) pectins at a low dose increased the frequencies of intestinal T-helper (Th)1 and Th2 cells after 1 week of pectin supplementation in mice, whereas DM18(low-DB) did not. After 4 weeks of supplementation with those pectins, Th1 and Th2 frequencies returned to control levels, whereas Rorγt+ regulatory T-cell frequencies increased. These structure-dependent effects could derive from induced shifts in microbiota composition that differed between DM18(low-DB) pectin and the other pectins. T-cell-modulating effects were not short-chain-fatty acid-dependent, but rather through an increase in Aryl-hydrocarbon-receptor-activating components. Thus, pectins with a specific combination of DM and DB have an impact on intestinal T cell-immunity in mice, when supplemented at a low dose.

The impact of the level and distribution of methyl-esters of pectins on TLR2-1 dependent anti-inflammatory responses.

Pectins have anti-inflammatory effects via Toll-like receptor (TLR) inhibition in a degree of methyl-esterification-(DM)-dependent manner. However, pectins also vary in distribution of methyl-esters over the galacturonic-acid (GalA) backbone (Degree of Blockiness - DB) and impact of this on anti-inflammatory capacity is unknown. Pectins mainly inhibit TLR2-1 but magnitude depends on both DM and DB. Low DM pectins (DM18/19) with both low (DB86) and high DB (DB94) strongly inhibit TLR2-1. However, pectins with intermediate DM (DM43/DM49) and high DB (DB60), but not with low DB (DB33), inhibit TLR2-1 as strongly as low DM. High DM pectins (DM84/88) with DB71 and DB91 do not inhibit TLR2-1 strongly. Pectin-binding to TLR2 was confirmed by capture-ELISA. In human macrophages, low DM and intermediate DM pectins with high DB inhibited TLR2-1 induced IL-6 secretion. Both high number and blockwise distribution of non-esterified GalA in pectins are responsible for the anti-inflammatory effects via inhibition of TLR2-1.

Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions.

BACKGROUND: The discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass. Through an oxidative mechanism, these enzymes are able to cleave and depolymerize various polysaccharides, acting not only on crystalline substrates such as chitin and cellulose, but also on other polysaccharides, such as xyloglucan, glucomannan and starch. Despite their widespread use, uncertainties related to substrate specificity and stereospecificity, the nature of the co-substrate, in-process stability, and the nature of the optimal reductant challenge their exploitation in biomass processing applications. RESULTS: In this work, we studied the properties of a novel fungal LPMO from the thermophilic fungus Thielavia australiensis, TausLPMO9B. Heterologous expression of TausLPMO9B in Aspergillus niger yielded a glycosylated protein with a methylated N-terminal histidine showing LPMO activity. High sequence identity of the AA9 domain to that of MtLPMO9B (MYCTH_80312) from Myceliophthora thermophila (84%) indicated strictly C1-oxidizing activity on cellulose, which was confirmed experimentally by the analysis of products released from cellulose using HPAEC. The enzyme was stable and active at a pH ranging from 4 to 6, thus matching the conditions commonly used in industrial biomass processing, where a low pH (between 4 and 5) is used due to the pH-optima of commercial cellulases and a desire to limit microbial contamination. CONCLUSION: While the oxidative cleavage of phosphoric acid swollen cellulose (PASC) by TausLPMO9B was boosted by the addition of H2O2 as a co-substrate, this effect was not observed during the saccharification of acid pretreated corn stover. This illustrates key differences between the lab-scale tests with artificial, lignin-free substrates and industrial settings with lignocellulosic biomass as substrate.

Sensing and transduction of nutritional and chemical signals in filamentous fungi: Impact on cell development and secondary metabolites biosynthesis.

Filamentous fungi respond to hundreds of nutritional, chemical and environmental signals that affect expression of primary metabolism and biosynthesis of secondary metabolites. These signals are sensed at the membrane level by G protein coupled receptors (GPCRs). GPCRs contain usually seven transmembrane domains, an external amino terminal fragment that interacts with the ligand, and an internal carboxy terminal end interacting with the intracellular G protein. There is a great variety of GPCRs in filamentous fungi involved in sensing of sugars, amino acids, cellulose, cell-wall components, sex pheromones, oxylipins, calcium ions and other ligands. Mechanisms of signal transduction at the membrane level by GPCRs are discussed, including the internalization and compartmentalisation of these sensor proteins. We have identified and analysed the GPCRs in the genome of Penicillium chrysogenum and compared them with GPCRs of several other filamentous fungi. We have found 66 GPCRs classified into 14 classes, depending on the ligand recognized by these proteins, including most previously proposed classes of GPCRs. We have found 66 putative GPCRs, representatives of twelve of the fourteen previously proposed classes of GPCRs, depending on the ligand recognized by these proteins. A staggering fortytwo putative members of the new GPCR class XIV, the so-called Pth11 sensors of cellulosic material as reported for Neurospora crassa and some other fungi, were identified. Several GPCRs sensing sex pheromones, known in yeast and in several fungi, were also identified in P. chrysogenum, confirming the recent unravelling of the hidden sexual capacity of this species. Other sensing mechanisms do not involve GPCRs, including the two-component systems (HKRR), the HOG signalling system and the PalH mediated pH transduction sensor. GPCR sensor proteins transmit their signals by interacting with intracellular heterotrimeric G proteins, that are well known in several fungi, including P. chrysogenum. These G proteins are inactive in the GDP containing heterotrimeric state, and become active by nucleotide exchange, allowing the separation of the heterotrimeric protein in active Gα and Gßγ dimer subunits. The conversion of GTP in GDP is mediated by the endogenous GTPase activity of the G proteins. Downstream of the ligand interaction, the activated Gα protein and also the Gß/Gγ dimer, transduce the signals through at least three different cascades: adenylate cyclase/cAMP, MAPK kinase, and phospholipase C mediated pathways.

Self-Regulated Plasma Heat Flux Mitigation Due to Liquid Sn Vapor Shielding.

A steady-state high-flux H or He plasma beam was balanced against the pressure of a Sn vapor cloud for the first time, resulting in a self-regulated heat flux intensity near the liquid surface. A temperature response of the liquid surface characterized by a decoupling from the received heating power and significant cooling of the plasma in the neutral Sn cloud were observed. The plasma heat flux impinging on the target was found to be mitigated, as heat was partially dissipated by volumetric processes in the vapor cloud rather than wholly by surface effects. These results motivate further exploration of liquid metal solutions to the critical challenge of heat and particle flux handling in fusion power plants.

Advanced Thomson scattering system for high-flux linear plasma generator.

An advanced Thomson scattering system has been built for a linear plasma generator for plasma surface interaction studies. The Thomson scattering system is based on a Nd:YAG laser operating at the second harmonic and a detection branch featuring a high etendue (f/3) transmission grating spectrometer equipped with an intensified charged coupled device camera. The system is able to measure electron density (n(e)) and temperature (T(e)) profiles close to the output of the plasma source and, at a distance of 1.25 m, just in front of a target. The detection system enables to measure 50 spatial channels of about 2 mm each, along a laser chord of 95 mm. By summing a total of 30 laser pulses (0.6 J, 10 Hz), an observational error of 3% in n(e) and 6% in T(e) (at n(e) = 9.4 × 10(18) m(-3)) can be obtained. Single pulse Thomson scattering measurements can be performed with the same accuracy for n(e) > 2.8 × 10(20) m(-3). The minimum measurable density and temperature are n(e) < 1 × 10(17) m(-3) and T(e) < 0.07 eV, respectively. In addition, using the Rayleigh peak, superimposed on the Thomson scattered spectrum, the neutral density (n(0)) of the plasma can be measured with an accuracy of 25% (at n(0) = 1 × 10(20) m(-3)). In this report, the performance of the Thomson scattering system will be shown along with unprecedented accurate Thomson-Rayleigh scattering measurements on a low-temperature argon plasma expansion into a low-pressure background.

Speciation despite globally overlapping distributions in Penicillium chrysogenum: the population genetics of Alexander Fleming's lucky fungus.

Eighty years ago, Alexander Fleming described the antibiotic effects of a fungus that had contaminated his bacterial culture, kick starting the antimicrobial revolution. The fungus was later ascribed to a putatively globally distributed asexual species, Penicillium chrysogenum. Recently, the species has been shown to be genetically diverse, and possess mating-type genes. Here, phylogenetic and population genetic analyses show that this apparently ubiquitous fungus is actually composed of at least two genetically distinct species with only slight differences detected in physiology. We found each species in air and dust samples collected in and around St Mary's Hospital where Fleming worked. Genotyping of 30 markers across the genome showed that preserved fungal material from Fleming's laboratory was nearly identical to derived strains currently in culture collections and in the same distinct species as a wild progenitor strain of current penicillin producing industrial strains rather than the type species P. chrysogenum. Global samples of the two most common species were found to possess mating-type genes in a near 1:1 ratio, and show evidence of recombination with little geographic population subdivision evident. However, no hybridization was detected between the species despite an estimated time of divergence of less than 1MYA. Growth studies showed significant interspecific inhibition by P. chrysogenum of the other common species, suggesting that competition may facilitate species maintenance despite globally overlapping distributions. Results highlight under-recognized diversity even among the best-known fungal groups and the potential for speciation despite overlapping distribution.

Intermediate frequency band digitized high dynamic range radiometer system for plasma diagnostics and real-time Tokamak control.

An intermediate frequency (IF) band digitizing radiometer system in the 100-200 GHz frequency range has been developed for Tokamak diagnostics and control, and other fields of research which require a high flexibility in frequency resolution combined with a large bandwidth and the retrieval of the full wave information of the mm-wave signals under investigation. The system is based on directly digitizing the IF band after down conversion. The enabling technology consists of a fast multi-giga sample analog to digital converter that has recently become available. Field programmable gate arrays (FPGA) are implemented to accomplish versatile real-time data analysis. A prototype system has been developed and tested and its performance has been compared with conventional electron cyclotron emission (ECE) spectrometer systems. On the TEXTOR Tokamak a proof of principle shows that ECE, together with high power injected and scattered radiation, becomes amenable to measurement by this device. In particular, its capability to measure the phase of coherent signals in the spectrum offers important advantages in diagnostics and control. One case developed in detail employs the FPGA in real-time fast Fourier transform (FFT) and additional signal processing. The major benefit of such a FFT-based system is the real-time trade-off that can be made between frequency and time resolution. For ECE diagnostics this corresponds to a flexible spatial resolution in the plasma, with potential application in smart sensing of plasma instabilities such as the neoclassical tearing mode (NTM) and sawtooth instabilities. The flexible resolution would allow for the measurement of the full mode content of plasma instabilities contained within the system bandwidth.

Development and testing of a fast Fourier transform high dynamic-range spectral diagnostics for millimeter wave characterization.

A fast Fourier transform (FFT) based wide range millimeter wave diagnostics for spectral characterization of scattered millimeter waves in plasmas has been successfully brought into operation. The scattered millimeter waves are heterodyne downconverted and directly digitized using a fast analog-digital converter and a compact peripheral component interconnect computer. Frequency spectra are obtained by FFT in the time domain of the intermediate frequency signal. The scattered millimeter waves are generated during high power electron cyclotron resonance heating experiments on the TEXTOR tokamak and demonstrate the performance of the diagnostics and, in particular, the usability of direct digitizing and Fourier transformation of millimeter wave signals. The diagnostics is able to acquire 4 GHz wide spectra of signals in the range of 136-140 GHz. The rate of spectra is tunable and has been tested between 200,000 spectra/s with a frequency resolution of 100 MHz and 120 spectra/s with a frequency resolution of 25 kHz. The respective dynamic ranges are 52 and 88 dB. Major benefits of the new diagnostics are a tunable time and frequency resolution due to postdetection, near-real time processing of the acquired data. This diagnostics has a wider application in astrophysics, earth observation, plasma physics, and molecular spectroscopy for the detection and analysis of millimeter wave radiation, providing high-resolution spectra at high temporal resolution and large dynamic range.

Construction of an hdfA Penicillium chrysogenum strain impaired in non-homologous end-joining and analysis of its potential for functional analysis studies.

The homologous recombination mechanism for DNA-repair is not predominant in most filamentous fungi, resulting in extremely low targeting efficiencies for molecular engineering. To increase the gene targeting efficiency, it is becoming common practice to inactivate the non-homologous end-joining (NHEJ) pathway that causes random integration, by deleting the fungal homologs of the human KU70 and KU80 genes that encode proteins functioning in the NHEJ pathway. This has been described for several filamentous fungi, but limited knowledge on the physiological consequences is available. In this study we characterized targeting efficiency and physiology of penicillinG producing Penicillium chrysogenum strains, in which the KU70 or KU80 homologues hdfA and hdfB had been deleted. Targeting efficiency was increased from ca. 1% in the reference strain to 47% and 56% in the hdfA and hdfB mutant strains, respectively, using an ends-out construct. Physiological and transcriptome analysis of glucose-limited chemostat cultures of the hdfA deletion strain and the reference strain showed minimal differences. Although, in a direct competition experiment to assess strain fitness, the reference strain had a clear advantage over the deletion strain, the results demonstrate the potential of DeltahdfAP. chrysogenum strains for the functional analysis of the recently completed P. chrysogenum genome sequence and in further metabolic engineering of antibiotics production.

A line-of-sight electron cyclotron emission receiver for electron cyclotron resonance heating feedback control of tearing modes.

An electron cyclotron emission (ECE) receiver inside the electron cyclotron resonance heating (ECRH) transmission line has been brought into operation. The ECE is extracted by placing a quartz plate acting as a Fabry-Perot interferometer under an angle inside the electron cyclotron wave (ECW) beam. ECE measurements are obtained during high power ECRH operation. This demonstrates the successful operation of the diagnostic and, in particular, a sufficient suppression of the gyrotron component preventing it from interfering with ECE measurements. When integrated into a feedback system for the control of plasma instabilities this line-of-sight ECE diagnostic removes the need to localize the instabilities in absolute coordinates.

Dietary patterns and their socio-demographic determinants in 10 European countries: data from the DAFNE databank.

OBJECTIVE: To describe the dietary patterns of 10 European countries and their socio-demographic determinants, using the comparable between-countries DAFNE data. DESIGN: Analysis of standardized and postharmonized data collected through the national household budget surveys. SETTING: Nationally representative surveys undertaken in 10 European countries, generally in the second half of the 1990s. RESULTS: The differences in the fruit and vegetable consumption previously identified between Mediterranean and Northern European countries seem to be leveling out, particularly in relation to fruit consumption. Pulses, however, still characterize the diet of the Mediterraneans. Straying from their traditional food choices, Mediterraneans recorded high availability of unprocessed red meat, while Central and Northern Europeans preferably consumed meat products. The household availability of beverages (alcoholic and non-alcoholic) is generally higher among Central and Northern European populations. Principal component (PC) analysis led to the identification of two dietary patterns in each of the 10 countries. The first was similar in all countries and indicated 'wide-range' food buyers. The second was slightly more varied and described 'beverage and convenience' food buyers. PC1 was common among households of retired and elderly members, while PC2 was common among households located in urban or semi-urban areas and among adult Scandinavians living alone. CONCLUSIONS: The dietary patterns identified point towards a progressive narrowing of dietary differences between North and South European countries. The comparable between-countries DAFNE data could prove useful in ecological studies, in the formulation of dietary guidelines and public health initiatives addressing specific population groups. SPONSORSHIP: European Commission.

Compartmentalization and transport in beta-lactam antibiotics biosynthesis.

Classical strain improvement of beta-lactam producing organisms by random mutagenesis has been a powerful tool during the last century. Current insights in the biochemistry and genetics of beta-lactam production, in particular in the filamentous fungus Penicillium chrysogenum, however, make a more directed and rational approach of metabolic pathway engineering possible. Besides the need for efficient genetic methods, a thorough understanding is needed of the metabolic fluxes in primary, intermediary and secondary metabolism. Controlling metabolic fluxes can be achieved by adjusting enzyme activities and metabolite levels in such a way that the main flow is directed towards the desired product. In addition, compartmentalization of specific parts of the beta-lactam biosynthesis pathways provides a way to control this pathway by clustering enzymes with their substrates inside specific membrane bound structures sequestered from the cytosol. This compartmentalization also requires specific membrane transport steps of which the details are currently uncovered.

LIBS using dual- and ultra-short laser pulses.

Pre-ablation dual-pulse LIBS enhancement data for copper, brass and steel using ns laser excitation are reported. Although large enhancements are observed for all samples, the magnitude of the enhancement is matrix dependent. Whereas all of the dual-pulse studies used ns laser excitation we see interesting effects when using ps and fs laser excitation for single-pulse LIBS. LIBS spectra of copper using 1.3 ps and 140 fs laser pulses show much lower background signals compared to ns pulse excitation. Also, the atomic emission decays much more rapidly with time. Because of relatively low backgrounds when using ps and fs pulses, non-gated detection of LIBS is shown to be very effective. The plasma dissipates quickly enough using ps and fs laser pulses, that high pulse rates, up to 1,000 Hz, are effective for increasing the LIBS signal, for a given measurement time. Finally, a simple near-collinear dual-pulse fiber-optic LIBS probe is shown to be useful for enhanced LIBS measurements.

Diverse deletions in the growth hormone receptor gene cause growth hormone insensitivity syndrome.

Growth hormone insensitivity syndrome (GHIS; also known as Laron syndrome), is characterized by severe postnatal growth failure and normal growth hormone. The syndrome is frequently caused by point mutations in the growth hormone receptor gene (GHR). Here we report five families with GHIS and partial deletions of the GHR gene. The deletion breakpoints were sequenced and PCR-based diagnostic tests were developed. In a Cambodian family, a novel deletion removed part of exon 5 and 1.2 kb of the preceding intron. The deletion occurred by recombination within four identical nucleotides. In the mutant transcript, skipping of the truncated exon 5 leads to a frameshift and premature termination codon (PTC). A previously reported discontinuous deletion of GHR exons 3, 5, and 6 was identified in three Oriental Jewish families. An unaffected individual was heterozygous for the exon 5 and 6 deletion, but homozygously deleted for exon 3 suggesting that the exon 3 deletion is a polymorphism. The pathogenic deletion of exons 5 and 6 spans about 7.5 kb. Sequence analysis of the breakpoints revealed an imperfect junction between introns 4 and 6, with a four basepair insertion. A novel deletion of 13 nucleotides within exon 9 was identified in a Caucasian girl with GHIS who carries the I153T missense mutation on her other allele. The exon 9 deletion leads to a frameshift and PTC. The predicted protein retains the transmembrane domain and a short cytoplasmic tail. Four family members in three generations were carriers of this deletion, but only two of them were below normal for height, suggesting that this mutation by itself does not act as a dominant negative, as was reported for two other GHR mutations which lead to truncation of the intracellular domain.

Concentration and memory deficits in patients with fibromyalgia syndrome.

The present study compared 30 patients with Fibromyalgia Syndrome (FS) to 30 healthy control subjects matched for age, sex, and estimated intellectual level on standardized measures of attention, concentration, and memory as well as subjective ratings of memory abilities and sleep quality. In addition, in order to investigate the relationship between cognitive functioning and other physical and psychological symptoms, subjects with FS completed psychological measures of pain severity, trait anxiety, and depression. Results indicated that patients with FS performed more poorly on tests of immediate and delayed recall, and sustained auditory concentration, and their ratings of both their memory abilities and sleep quality were lower than those of controls. Furthermore, perceived memory deficits of the FS subjects were disproportionately greater than their objective deficits. Results indicated significant correlations between performance on memory and concentration measures and scores on questionnaires of pain severity and trait anxiety. Implications of these results for multidisciplinary treatment programs are discussed.

Supermarket sales data: a tool for measuring regional differences in dietary habits.

OBJECTIVE: To assess how well supermarket sales data from a major supermarket chain can reflect on regional differences in dietary behaviour by comparing the sales data with the results provided by the annual health behaviour surveys. DESIGN: Cross-sectional observational study. SETTING: The study was carried out in six Finnish cities situated in different parts of Finland. For the study supermarket sales data of milk, sour milk, fats and oils for 1 month, September 1997, were obtained from eight supermarkets. Proportional sales of different types of dairy products were calculated as well as mean salt and fat per cent and the proportion of saturated fat to total fat. The health behaviour surveys from spring 1995, 1996 and 1997 provided information about dietary habits of the adult population in the cities. The reported use of dairy products was compared with the percentage sales. RESULTS: The proportional sales of dairy products varied between the cities. In Pori in western Finland the sale of milk fat was highest in all food groups. In Oulu, northern Finland, the sale of non-fat milk was high. In the capital region the sale of oil was highest. Regional differences could also be seen in the survey data. The similarity between the two different datasets was high. CONCLUSIONS: The use of supermarket sales data for assessing regional differences in health behaviour is feasible. The challenge will be to get supermarket managers willing to provide sales data on a routine basis for monitoring and research.

Engineering aspects of beta-lactam biosynthesis.

The adaptation of an existing industrial production line to boost production capacity (a hardware analogue of strain improvement) or to enable the production of a derivative of the original product is used as an example to explore the similarities between process design and engineering and metabolic pathway engineering. In the two fields the same principles appear to apply: for most process engineering solutions metabolic pathway engineering analogues can be found. Analogues are illustrated by reference to literature (e.g. overproduction of amino acid precursors at the 'bottom' of the pathway, relocalisation of proteins and overexpression of genes from the secondary metabolism). Some possible future applications are defined (e.g. supervisory manipulations as regulation by gene transcription factors).

Four contiguous amino acid substitutions, identified in patients with Laron syndrome, differently affect the binding affinity and intracellular trafficking of the growth hormone receptor.

We have analyzed the GH receptor (GHR) gene in four individuals with Laron syndrome, and a missense mutation was identified for each patient in the extracellular domain of the GHR (D152H, I153T, Q154P, and V155G). The D152H mutation was previously reported. We have reproduced the three novel mutations in the GHR complementary DNA and analyzed their consequences in human 293 transfected cells. In cells expressing the I153T and V155G mutants, binding of [125I]human GH at the cell surface was very low, whereas binding to total membrane fractions was much less affected, suggesting impaired cell surface expression. Binding assays with cells expressing the Q154P mutant revealed severe defects both at the cell surface and in total particulate membrane fractions. Immunofluorescence experiments confirmed that cell surface expression of the three mutants was altered, and colocalization studies suggested that most of the mutant receptors are retained in the endoplasmic reticulum. Endoglycosidase H resistance tests also indicated that the majority of I153T and V155G GHRs are trapped in the endoplasmic reticulum. Thus, mutations on contiguous amino acids of the GHR result in various defects. The I153T, Q154P, and V155G mutations mainly affect intracellular trafficking and binding affinity of the receptor, whereas the D152H mutation affects receptor expression, dimerization, and signaling.