Search

How the number of genome copies modifies the effect of random mutations remains poorly known. In yeast, researchers have investigated these effects for knock-out or other large-effect mutations, but have not accounted for differences at the mating-type locus. We set out to compare fitness differences among strains that differ in ploidy and/or zygosity using a panel of spontaneously arising mutations acquired in haploid yeast from a previous study. To ensure no genetic differences, even at the mating-type locus, we embarked on a series of transformations, which first sterilized and then temporarily introduced plasmid-borne mating types. Despite these attempts to equalize the haplotypes, fitness variation introduced during transformation swamped the differences among the original mutation-accumulation lines. While colony size looked normal, we observed a bi-modality in the maximum growth rate of our transformed yeast and determined that many of the slow growing lines were respiratory deficient ("petite"). Not previously reported, we found that yeast that were TID1/RDH54 knockouts were less likely to become petite. Even for lines with the same petite status, however, we found no correlation in fitness between the two replicate transformations performed. These results pose a challenge for any study using transformation to measure the fitness effect of genetic differences among strains. By attempting to hold haplotypes constant, we introduced more mutations that overwhelmed our ability to measure fitness differences between the genetic states. In this study, we transformed over one hundred different lines of yeast, using two independent transformations, and found that this common laboratory procedure can cause large changes to the microbe studied. Our study provides a cautionary tale of the need to use multiple transformants in fitness assays. [ABSTRACT FROM AUTHOR]

Cyst producing nematodes are persistent soil-borne organisms causing severe damage to cultivated plants. Persistence of the economically relevant cyst nematode species Globodera pallida, G. rostochiensis and Heterodera schachtii was investigated at different stages during a large-scale industrial composting process to evaluate its efficiency to prevent spread of these nematodes into natural and agricultural habitats. Using reference cyst nematodes incorporated into organic waste from households and the processing industry, the effect of anaerobic fermentation and aerobic composting processes were investigated. Treated cysts were analysed for viability and reproductive potential by performing hatching tests and bioassays on susceptible host plants. The investigated composting plant showed temperatures between 40 and 72 °C at aerobic composting conditions depending on the sample position (bottom, middle, top) within the pile. We found no viable juveniles or reproductive potential of Globodera spp. (Loof and Bakker 1992) and less than 5 per cent reproduction of H. schachtii. Additionally to temperature conditions, we presume that competition of the microbial community and their released biodigestants like fatty acids also play a major role in successful treatment of these severe pest organisms. [ABSTRACT FROM AUTHOR]

The dissemination of soil tares in the potato and sugar beet processing industry is one of the main paths for the spread of potato cyst nematodes (PCN), a severe quarantine pest. Efficient measures for the disinfestation of tare soil from PCN, but also from beet cyst nematodes (BCN), are needed. In our study, Globodera pallida (a PCN) and Heterodera schachtii (a BCN) cysts were sealed in gauze bags and imbedded in sedimentation basins. The cysts were either placed (a) in a presedimentation basin (Brukner basin) for three days, (b) in the presedimentation basin for three days and subsequently in sedimentation basins for nine weeks or (c) in sedimentation basins for nine weeks (without presedimentation). We tested the viability of the eggs and juveniles by hatching assays and using the reproduction rates in bioassays. We demonstrated that PCN and BCN imbedded in a sedimentation basin were only still showing some hatching activity after 2.5 weeks, while no hatching was observed when an additional Brukner basin treatment was conducted before sedimentation. [ABSTRACT FROM AUTHOR]

Scientific and technological advances within the life sciences have enabled the generation of very large datasets that must be processed, stored, and managed computationally. Researchers increasingly require data science skills to work with these datasets at scale in order to convert information into actionable insights, and undergraduate educators have started to adapt pedagogies to fulfill this need. Course-based undergraduate research experiences (CUREs) have emerged as a leading model for providing large numbers of students with authentic research experiences including data science. Originally designed around wetlab research experiences, CURE models have proliferated and diversified globally to accommodate a broad range of academic disciplines. Within microbiology, diversity metrics derived from microbiome sequence information have become standard data products in research. In some cases, researchers have deposited data in publicly accessible repositories, providing opportunities for reproducibility and comparative analysis. In 2020, with the onset of the COVID-19 pandemic and concomitant shift to remote learning, the University of British Columbia set out to develop an online data science CURE in microbiology. A team of faculty with collective domain expertise in microbiome research and CUREs developed and implemented a data science CURE in which teams of students learn to work with large publicly available datasets, develop and execute a novel scientific research project, and disseminate their findings in the online Undergraduate Journal of Experimental Microbiology and Immunology. Analysis of the resulting studentauthored research articles, including comments from peer reviews conducted by subject matter experts, demonstrate high levels of learning effectiveness. Here, we describe core insights from course development and implementation based on a reverse course design model. Our approach to course design may be applicable to the development of other data science CUREs. [ABSTRACT FROM AUTHOR]

Ceratitis capitata (Wiedemann) is a polyphagous plant pest organism causing serious damage on more than 300 fruit types. Although the medfly is present in Europe, it is so far not established in Germany. In the years 2015–2017, an official survey was conducted by the German Federal Plant Protection Organisations to elucidate the current occurrence of this organism in Germany. While in 2015 only small numbers of flies were captured in traps situated in all German Federal States, a peak of 188 specimens could be trapped in 2016 with a decline to 29 specimens in 2017. Highest abundance of the fruit fly was detected in Southern Germany and at a single spot east of Berlin. The present study aimed to identify the origin of the trapped fruit flies and to assess the general potential of the species for overwintering in Germany. Using two approaches with cytochrome oxidase subunit I (COI), Sanger barcoding sequencing first and subsequent analysis of ten small simple repeat microsatellite loci from literature, the worldwide spread of C. capitata describing three phases could be retraced by analysing samples from Germany and 11 other countries. German and French populations were found to build the most recent stage of this species distribution. Specimens trapped in Germany obviously resulted from different introduction events. They most strongly resembled populations from France and Croatia but were not identical to these populations. We found strong evidence that specimens captured between 2016 and 2017 could not overwinter in Germany. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Cultivated Plants / Journal für Kulturpflanzen is the property of Verlag Eugen Ulmer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Anoplophora glabripennis (Motschulsky 1853) (Coleoptera: Cerambycidae), the Asian Longhorned Beetle, is native to temperate and subtropical areas of China and the Korean peninsula. Due to its wide range of host plants, it is considered among the most economically important invasive plant pests. The morphological identification of A. glabripennis larvae can be confirmed by DNA barcoding, but obtaining the specimens from infested trees can be a demanding and challenging task. Therefore, non-invasive diagnostic tools based on DNA extracted from frass samples can be of key importance in phytosanitary surveys. In this study, an in silico generated real-time quantitative PCR test was developed for the detection of A. glabripennis DNA from frass material, which is naturally extruded from larval tunnels through cracks in the bark. Specificity was confirmed against a wide range of other wood-boring insect species frequently encountered during phytosanitary surveys and inclusivity was demonstrated for different populations of A. glabripennis from all main European outbreak areas. The test proved sensitive and reliable in detecting A. glabripennis DNA extracted from woody frass material of Acer saccharinum and Aesculus hippocastanum at least up to the 100-fold dilution. Furthermore, the test allowed the molecular identification of any life stage of the insect, including eggs and young larvae, whose morphological identification is impossible or very challenging. This study provides a reliable and sensitive molecular tool to detect A. glabripennis DNA in woody frass material, thus allowing a non-invasive sampling approach. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Cultivated Plants / Journal für Kulturpflanzen is the property of Verlag Eugen Ulmer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In plant pest diagnosis, Sanger sequencing of marker genes (DNA-barcoding) is the most applied and appropriate method for the identification of insects. Standard PM7/129 of the European and Mediterranean Plant Protection Organization (EPPO) includes a number of primers and PCR protocols for diagnosing insect pests. LCO1490 and HCO2198 primers recommended herein were shown to be excellent tools for amplifying a fragment of the COI gene from a vast range of arthropods. The COI barcoding region is available for thousands of arthropod taxa in public databases and ready-to-use for evolutionary studies. However, we found that LCO1490 and HCO2198 primers are not working for bark beetles of genus Ips. The attempt to amplify this gene fragment from an individual organism using the barcoding primers led to DNA amplification of associated wasps and nematodes, which were apparently vectored by the beetle. Thus, new primers for Ips that bind specifically to another (non-barcoding) region of the COI gene were developed in the past years. These primers were successfully applied in phylogenetic analyses of this genus, resulting in the adverse effect that COI-based Ips phylogenies cannot be expanded to higher systematic categories without sequencing the outgroups (as they are not available in databases yet). Here we provide new primers for Ips that differ significantly from DNA sequences of Ips-associated wasps and nematodes and bind to a COI fragment that largely overlaps with the barcoding region proposed in the EPPO standard. Furthermore, using these primers we developed a quick PCR-based test for detecting Ips duplicatus, a quarantine pest currently emerging in many European countries. [ABSTRACT FROM AUTHOR]

We live in an increasingly data-driven world, where high-throughput sequencing and mass spectrometry platforms are transforming biology into an information science. This has shifted major challenges in biological research from data generation and processing to interpretation and knowledge translation. However, postsecondary training in bioinformatics, or more generally data science for life scientists, lags behind current demand. In particular, development of accessible, undergraduate data science curricula has the potential to improve research and learning outcomes as well as better prepare students in the life sciences to thrive in public and private sector careers. Here, we describe the Experiential Data science for Undergraduate Cross-Disciplinary Education (EDUCE) initiative, which aims to progressively build data science competency across several years of integrated practice. Through EDUCE, students complete data science modules integrated into required and elective courses augmented with coordinated cocurricular activities. The EDUCE initiative draws on a community of practice consisting of teaching assistants (TAs), postdocs, instructors, and research faculty from multiple disciplines to overcome several reported barriers to data science for life scientists, including instructor capacity, student prior knowledge, and relevance to discipline-specific problems. Preliminary survey results indicate that even a single module improves student self-reported interest and/or experience in bioinformatics and computer science. Thus, EDUCE provides a flexible and extensible active learning framework for integration of data science curriculum into undergraduate courses and programs across the life sciences. [ABSTRACT FROM AUTHOR]

In multicellular organisms with specialized cells, the most significant distinction among cell types is between reproductive (germ) cells and non-reproductive/somatic cells (soma). Although soma contributed to the marked increase in complexity of many multicellular lineages, little is known about its evolutionary origins. We have previously suggested that the evolution of genes responsible for the differentiation of somatic cells involved the co-option of life history trade-off genes that in unicellular organisms enhanced survival at a cost to immediate reproduction. In the multicellular green alga, Volvox carteri, cell fate is established early in development by the differential expression of a master regulatory gene known as regA. A closely related RegA-Like Sequence (RLS1) is present in its single-celled relative, Chlamydomonas reinhardtii. RLS1 is expressed in response to stress, and we proposed that an environmentally induced RLS1-like gene was co-opted into a developmental pathway in the lineage leading to V. carteri. However, the exact evolutionary scenario responsible for the postulated cooption event remains to be determined. Here, we show that in addition to being developmentally regulated, regA can also be induced by environmental cues, indicating that regA has maintained its ancestral regulation. We also found that the absence of a functional RegA protein confers increased sensitivity to stress, consistent with RegA having a direct or indirect role in stress responses. Overall, this study (i) provides mechanistic evidence for the co-option of an environmentally induced gene into a major developmental regulator, (ii) supports the view that major morphological innovations can evolve via regulatory changes and (iii) argues for the role of stress in the evolution of multicellular complexity. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Cultivated Plants / Journal für Kulturpflanzen is the property of Verlag Eugen Ulmer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Cultivated Plants / Journal für Kulturpflanzen is the property of Verlag Eugen Ulmer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Cultivated Plants / Journal für Kulturpflanzen is the property of Verlag Eugen Ulmer and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Negatively charged tin clusters offer a broad range of decay products, as observed after electron-cluster interactions [S. König, M. Wolfram, S. Bandelow, G. Marx, L. Schweikhard, Eur. Phys. J. D 72, 153 (2018)]. To get further insight into their decay pathways, size and charge-state selected clusters Snn- and Snn2- were photo-excited at the ClusterTrap setup by 532 nm Nd:YAG laser pulses. For small mono-anionic precursor clusters containing up to n ≃ 45 atoms, Snn-7- and Snn-10- are observed as preferred ionic fragments. For bigger clusters a transition to neutral monomer evaporation was found. In the case of di-anionic precursors, preferred decay products are Sn10- and Snn-10-, indicating fission similar to the case of the group 14 neighbor element lead [S. König, A. Jankowski, G. Marx, L. Schweikhard, M. Wolfram, Phys. Rev. Lett. 120, 163001 (2018)]. Furthermore, doubly charged fragment clusters such as Snn-72- are observed, originating from break-off of neutral heptamers Sn7, a behavior which has not been observed previously for di-anionic clusters. [ABSTRACT FROM AUTHOR]

Selenium (Se) is an important micronutrient but also a strong toxin with a narrow tolerance range for many organisms. As such, a globally heterogeneous Se distribution in soils is responsible for various disease patterns (i.e. Se excess and deficiency) and environmental problems, whereby plants play a key role for the Se entrance into the biosphere. Selenium isotope variations were proved to be a powerful tracer for redox processes and are therefore promising for the exploration of the species dependent Se metabolism in plants and the Se cycling within the Critical Zone. Plant cultivation setups enable systematic controlled investigations, but samples derived from them–plant tissue and phytoagar–are particularly challenging and require specific preparation and purification steps to ensure precise and valid Se isotope analytics performed with HG-MC-ICP-MS. In this study, different methods for the entire process from solid tissue preparation to Se isotope measurements were tested, optimized and validated. A particular microwave digestion procedure for plant tissue and a vacuum filtration method for phytoagar led to full Se recoveries, whereby unfavorable organic residues were reduced to a minimum. Three purification methods predominantly described in the literature were systematically tested with pure Se solution, high concentrated multi-element standard solution as well as plant and phytoagar as target matrices. All these methods efficiently remove critical matrix elements, but differ in Se recovery and organic residues. Validation tests doping Se-free plant material and phytoagar with a reference material of known Se isotope composition revealed the high impact of organic residues on the accuracy of MC-ICP-MS measurements. Only the purification method with no detectable organic residues, hydride generation and trapping, results in valid mass bias correction for plant samples with an average deviation to true δ82/76Se values of 0.2 ‰ and a reproducibility (2 SD) of ± 0.2 ‰. For phytoagar this test yields a higher deviation of 1.1 ‰ from the true value and a 2 SD of ± 0.1 ‰. The application of the developed methods to cultivated plants shows sufficient accuracy and precision and is a promising approach to resolve plant internal Se isotope fractionations, for which respective δ82/76Se values of +2.3 to +3.5 ‰ for selenate and +1.2 to +1.9 ‰ for selenite were obtained. [ABSTRACT FROM AUTHOR]

Abstract: The redox‐sensitive, chalcophile, and volatile Se stable isotope system offers new perspectives to investigate the origin and evolution of terrestrial volatiles and the roles of magmatic and recycling processes in the development of the redox contrast between Earth's reservoirs. Selenium isotope systematics become more robust in a well‐constrained petrogenetic context as can be inferred from Se‐Te elemental signatures of sulfides and igneous rocks. In this study, we present a high‐yield chemical sample processing method that allows the determination of Se‐Te concentrations and Se isotope composition from the same sample digest of silicate rocks by hydride generation isotope dilution (ID) quadrupole inductively coupled plasma mass spectrometry (ICP‐MS) and double spike (DS) multicollector (MC)‐ICP‐MS, respectively. Our procedure yields ∼80% Se‐Te recoveries with quantitative separation of relevant interfering elements such as Ge and HG‐buffering metals. Replicate analyses of selected international reference materials yield uncertainties better than 0.11‰ (2 s.d.) on δ82/76Se and 3% (r.s.d.) on Se concentration for DS MC‐ICP‐MS determinations for as low as ∼10 ng sample Se. The precision of Se‐Te concentration measurements by ID ICP‐MS is better than 3% and 5% (r.s.d.) for total amounts of ∼0.5–1 ng Se and ∼0.2–0.5 ng Te, respectively. The basaltic reference materials have variable Se‐Te contents, but their δ82/76Se values are rather uniform (on average 0.23 ± 0.14‰; 2 s.d.) and different from the chondritic value. This altogether provides the methodology and potential to extend the limited data set of coupled Se isotope and Se‐Te elemental systematics of samples relevant to study the terrestrial igneous inventory. [ABSTRACT FROM AUTHOR]

Fungi are prominent drivers of ecological processes in soils, so that fungal communities across different soil ecosystems have been well investigated. However, for arable soils taxonomically resolved fine-scale studies including vertical itemization of fungal communities are still missing. Here, we combined a cloning/Sanger sequencing approach of the ITS/LSU region as marker for general fungi and of the partial SSU region for arbuscular mycorrhizal fungi (AMF) to characterize the microbiome in different maize soil habitats. Four compartments were analyzed over two annual cycles 2009 and 2010: a) ploughed soil in 0–10 cm, b) rooted soil in 40–50 cm, c) root-free soil in 60–70 cm soil depth and d) maize roots. Ascomycota was the most dominant phylum across all compartments. Fungal communities including yeasts and AMF differed strongly between compartments. Inter alia, Tetracladium, the overall largest MOTU (molecular operational taxonomic unit), occurred in all compartments, whereas Trichosporon dominated all soil compartments. Sequences belonging to unclassified Helotiales were forming the most abundant MOTUs exclusively present in roots. This study gives new insights on spatial distribution of fungi and helps to link fungal communities to specific ecological properties such as varying resources, which characterize particular niches of the heterogeneous soil environment. [ABSTRACT FROM AUTHOR]

We report on a lab-on-a-chip approach for on-site detection of Phytophthora species that allows visual signal readout. The results demonstrate the significance of single-stranded DNA (ssDNA) generation in terms of improving the intensity of the hybridization signal and to improve the reliability of the method. Conventional PCR with subsequent heat denaturation, sodium hydroxide-based denaturation, lambda exonuclease digestion and two asymmetric PCR methods were investigated for the species P. fragariae, P. kernoviae, and P. ramorum. The positioning of the capture probe within the amplified yeast GTP-binding protein (YPT1) target DNA was also of interest because it significantly influences the intensity of the signal. Statistical tests were used to validate the impact of the ssDNA generation methods and the capture-target probe position. The single-stranded target DNA generated by Linear-After-The-Exponential PCR (LATE-PCR) was found to produce signal intensities comparable to post-PCR exonuclease treatment. The LATE-PCR is the best method for the on-site detection of Phytophthora because the enzymatic digestion after PCR is more laborious and time-consuming. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

As a severe danger to potato plants, potato cyst nematodes (PCNs) are regulated quarantine pests in many countries. Adhering soils to potato or other tuber crops provoke the uncontrolled spread of the pest after their industrial processing. The disinfection of soil tares to inactivate PCNs is therefore compulsory. In the present study, we test whether the viability and formation of PCN cysts are suppressed after treatment with γ and β irradiation. In a first experiment, nematode cysts were treated with γ or β irradiation without soil matrix to determine the minimum dose parameters in a range between 0–12 kGy. Subsequently, two representative soils that included cysts were irradiated. Irradiation effects on PCN viability and the formation of new cysts were evaluated via hatching tests and bioassays with susceptible potato plants, respectively. In addition, maize seedling growth was tested in irradiated soil tares. We found that next to γ also, β irradiation suppressed the hatch of PCN juveniles and its formation of cysts when placed in soil tares. The growth of maize seedlings was not impaired in irradiated soils compared to control soils. Both γ and β irradiation treatment offer an organism group selective and effective phytosanitary measures to disinfect soil tares from PCNs. [ABSTRACT FROM AUTHOR]

Recent studies assessing the role of biological diversity for ecosystem functioning indicate that the diversity of functional traits and the evolutionary history of species in a community, not the number of taxonomic units, ultimately drives the biodiversity-ecosystem-function relationship. Here, we simultaneously assessed the importance of plant functional trait and phylogenetic diversity as predictors of major trophic groups of soil biota (abundance and diversity), six years from the onset of a grassland biodiversity experiment. Plant functional and phylogenetic diversity were generally better predictors of soil biota than the traditionally used species or functional group richness. Functional diversity was a reliable predictor for most biota, with the exception of soil microorganisms, which were better predicted by phylogenetic diversity. These results provide empirical support for the idea that the diversity of plant functional traits and the diversity of evolutionary lineages in a community are important for maintaining higher abundances and diversity of soil communities. [ABSTRACT FROM AUTHOR]

Decomposers and arbuscular mycorrhizal fungi (AMF) both determine plant nutrition; however, little is known about their interactive effects on plant communities. We set up a greenhouse experiment to study effects of plant competition (one- and two-species treatments), Collembola ( Heteromurus nitidus and Protaphorura armata), and AMF ( Glomus intraradices) on the performance (above- and belowground productivity and nutrient uptake) of three grassland plant species ( Lolium perenne, Trifolium pratense, and Plantago lanceolata) belonging to three dominant plant functional groups (grasses, legumes, and herbs). Generally, L. perenne benefited from being released from intraspecific competition in the presence of T. pratense and P. lanceolata. However, the presence of AMF increased the competitive strength of P. lanceolata and T. pratense against L. perenne and also modified the effects of Collembola on plant productivity. The colonization of roots by AMF was reduced in treatments with two plant species suggesting that plant infection by AMF was modified by interspecific plant interactions. Collembola did not affect total colonization of roots by AMF, but increased the number of mycorrhizal vesicles in P. lanceolata. AMF and Collembola both enhanced the amount of N and P in plant shoot tissue, but impacts of Collembola were less pronounced in the presence of AMF. Overall, the results suggest that, by differentially affecting the nutrient acquisition and performance of plant species, AMF and Collembola interactively modify plant competition and shape the composition of grassland plant communities. The results suggest that mechanisms shaping plant community composition can only be understood when complex belowground interactions are considered. [ABSTRACT FROM AUTHOR]

Pyruvate decarboxylase is a key enzyme in organisms whose energy metabolism is based on alcoholic fermentation. The enzyme catalyses the nonoxidative decarboxylation of 2-oxo acids in the presence of the cofactors thiamine diphosphate and magnesium ions. Pyruvate decarboxylase species from yeasts and plant seeds studied to date are allosterically activated by their substrate pyruvate. However, detailed kinetic studies on the enzyme from Neurospora crassa demonstrate for the first time the lack of substrate activation for a yeast pyruvate decarboxylase species. The quaternary structure of this enzyme species is also peculiar because it forms filamentous structures. The complex enzyme structure was analysed using a number of methods, including small-angle X-ray solution scattering, transmission electron microscopy, analytical ultracentrifugation and size-exclusion chromatography. These measurements were complemented by detailed kinetic studies in dependence on the pH. Structured digital abstract [ABSTRACT FROM AUTHOR]

The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5′-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5′-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites. Structured digital abstract • : EcODC (uniprotkb: ) and EcODC (uniprotkb: ) bind ( ) by X-ray scattering ( ) • : EcODC (uniprotkb: ) and EcODC (uniprotkb: ) bind ( ) by X-ray crystallography ( ) [ABSTRACT FROM AUTHOR]

Measurement of heart rate variability (HRV) shows information on the functional state of the autonomic nervous system (ANS). In adults there are standardized autonomic tests and well-established ranges of normal values, which is not the case in children. The aim of the present study was (1) to introduce an ANS test battery, especially for children and adolescents; (2) to establish normative HRV parameters; and (3) to determine the impact of ANS tests on HRV parameters compared with baseline measurements. We investigated 100 healthy children and adolescents between 6 and 15 years old. We subdivided the investigated group into a group of children (5–11 years old) and adolescents (12–15 years old) and measured HRV by time and frequency domain parameters during baseline, rhythmic breathing, Valsalva test, active standing, tilt-table testing, and handgrip test. The normative HRV data are presented by means, SDs, medians, and percentiles. The results described refer to baseline values for each HRV parameter separately to demonstrate the influence of age on HRV parameters. The study results present not only first normative HRV data for an autonomic test battery especially adapted to children and adolescents, but they also quantify the autonomic changes induced by test procedures compared with baseline measurements. [ABSTRACT FROM AUTHOR]

ALTSim is a MATLAB-based simulator of several associative learning models, including Pearce's configural model, the extended configural model, the Rescorla--Wagner model, the unique cue hypothesis, the modified unique cue hypothesis, the replaced elements model, and Harris's elemental model. It allows for specifying all relevant parameters, as well as exact stimulus sequences by graphical user interfaces. It is an easy-to-use tool that facilitates evaluating and comparing the featured associative learning models. ALTSim is available free of charge from www.staff.uni-marburg.de/~lachnit/ALTSim/. [ABSTRACT FROM AUTHOR]

Phospholipases D play an important role in the regulation of cellular processes in plants and mammals. Moreover, they are an essential tool in the synthesis of phospholipids and phospholipid analogs. Knowledge of phospholipase D structures, however, is widely restricted to sequence data. The only known tertiary structure of a microbial phospholipase D cannot be generalized to eukaryotic phospholipases D. In this study, the isoenzyme form of phospholipase D from white cabbage (PLDα2), which is the most widely used plant phospholipase D in biocatalytic applications, has been characterized by small-angle X-ray scattering, UV-absorption, CD and fluorescence spectroscopy to yield the first insights into its secondary and tertiary structure. The structural model derived from small-angle X-ray scattering measurements reveals a barrel-shaped monomer with loosely structured tops. The far-UV CD-spectroscopic data indicate the presence of α-helical as well as β-structural elements, with the latter being dominant. The fluorescence and near-UV CD spectra point to tight packing of the aromatic residues in the core of the protein. From the near-UV CD signals and activity data as a function of the calcium ion concentration, two binding events characterized by dissociation constants in the ranges of 0.1 mm and 10–20 mm can be confirmed. The stability of PLDα2 proved to be substantially reduced in the presence of calcium ions, with salt-induced aggregation being the main reason for irreversible inactivation. [ABSTRACT FROM AUTHOR]

Stimuli presented with a low frequency elicit larger pupillary responses than those presented with a high frequency. Similar effects are known for motor responses observed in reaction time experiments. Utilizing this stimulus probability effect, we conducted a Go/NoGo reaction time experiment and measured pupillary dilation to evaluate categorization. We found no probability effect when participants used distinctive features to classify stimuli with different frequencies into the same category. If no distinctive features could be applied, however, the effect was observed in both pupillary and reaction time data. The stimulus probability effect can serve as a tool for the evaluation of categorization and is discussed in the context of orienting. [ABSTRACT FROM AUTHOR]

Acute pancreatitis is rarely seen in children, and, in contrast to cases in adults, it is often drug induced. One possible medication is the antiepileptic drug valproic acid (VPA), which is commonly prescribed for generalized and focal epilepsy, migraine, neuropathic pain, and bipolar disorder. The common side effects associated with VPA are typically benign, but less common but more serious adverse effects may occur. These include hepatotoxicity, hyperammonemic encephalopathy, coagulation disorders, and pancreatitis. Since 1979, a few cases of pancreatitis induced by VPA have been published in the medical literature. We mailed a questionnaire to all members of the “German Section of the International League against Epilepsy,” asking about VPA-induced side effects. We also reviewed the medical literature for VPA-induced pancreatitis. Fifty-three publications (90 patients) published from 1979 to 2005 were found. Our survey in Germany, however, yielded 16 cases of pancreatitis from 1994 to 2003 whose original files we could study in detail. None of these patients had been published previously. The difference between 90 patients reported worldwide from 1979 to 2005 and the 16 new documented cases from only Germany over 10 years corroborates that the occurrence of this severe side effect is under reported. [ABSTRACT FROM AUTHOR]

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 Å resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106–113 and 292–301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105–113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105–113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

Purpose: Valproic acid (VPA) is an antiepileptic drug (AED) commonly used for generalized and focal epilepsies. The clinical relevance of coagulopathies, known as side effects of VPA therapy, especially thrombocytopenia, von Willebrand disease, and a decrease of factor XIII, is still unclear. Methods: In our institute, we noticed a high incidence of clinically relevant coagulation problems related to VPA in eight patients within 1 year only and a further seven children with significant coagulopathy were identified in the context of planned surgery. Results: We provide an overview of these patients and all six VPA-induced coagulopathies. Conclusions: At this time, it cannot be recommended to control all hemostatic parameters in every patient. Whenever an increased bleeding tendency is observed, or before surgical procedures, a platelet count, thrombelastography, prothrombin time, activated partial thromboplastin time, TT, fibrinogen, von Willibrand factor, and factor XIII should be examined. With 385 VPAtreated patients per year and 15 cases of coagulation disorders in this period, we estimate the incidence of coagulation disorders related to VPA in children to be nearly 4%. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate whether the anticipatory pupillary dilation response is a useful indicator for the examination of complex differential conditioning problems like patterning. A human fear conditioning procedure with six groups ( n=20 each) was used to examine conditioned stimulus (CS) processing when a compound stimulus was reinforced, but not its elements (positive patterning) or when the elements were reinforced, but not the compound (negative patterning), as well as modifications in which the compound was replaced by either a new compound or by a new element. We found evidence for conditioning within 2 s after CS onset. Group differences in differential conditioning indicated systematic differences in CS processing due to different discrimination task difficulties. This study showed that the pupil response is a suitable indicator for human Pavlovian conditioning with high time resolution within and between trials. [ABSTRACT FROM AUTHOR]

Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4′-methylamino-thiamin diphosphate and N1′-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4′-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1′-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1′-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme[Lindqvist Y, Schneider G, Ermler U, Sundstrøm M (1992)EMBO J11, 2373–2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1′-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H/D-exchange at C2. Methylation at the N1′ position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H/D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of theα-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate. [ABSTRACT FROM AUTHOR]

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 Å resolution and refined to a crystallographic R-factor of 20.5% (R free 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open α/β topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis , but with a relative difference of 20° in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan. [ABSTRACT FROM AUTHOR]

Enterobacter cloacae , isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al . [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem. 16, 1207–1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase. [ABSTRACT FROM AUTHOR]

Summary: Purpose: To investigate a possible relation between antiepileptic drugs (AEDs) and the fatty acid composition of membranes. Methods: Fatty acid (FA) composition of erythrocytes was studied in children with epilepsy receiving AED monotherapy. Children taking valproate (VPA, n = 28), carbamazepine (CBZ, n = 17), or phenobarbitone (PB, n = 14) were compared with healthy controls (n = 25). FAs were measured by capillary-gas chromatography (GC-FID). Results: Significant changes (p < 0.05) in the FA composition of membranes were found. In children treated with VPA, C13:0 was decreased (8.2 ± 2% vs. 10.7 ± 4% in controls) and C14:0 increased (1.4 ± 0.5% vs.1 ± 0.5% in controls). C17:0 again was reduced (9.9 ± 4% vs. 13.2 ± 6% in controls), whereas the long-chained acids were enhanced: C18:2n-6 (6 ± 2.4% vs. 3.9 ± 2.5% in controls), and C20:4n-6 (1.9 ± 1.7% vs. 1.4 ± 0.5% in controls). The nonidentified FAs also increased with VPA therapy: 2.5 ± 0.8% versus 1.7 ± 0.9% in controls. Children treated with CBZ showed only minimal changes of FA composition: C13:0 was decreased compared with controls (8 ± 2% vs. 10.7 ± 4%). No changes were seen in patients taking PB. The mean corpuscular volume (MCV) showed important differences between the study groups: MCV was 84.7 ± 6.0 fl during VPA therapy (p < 0.001) and 85.7 ± 4.1 fl with CBZ (p < 0.001). During PB, the MCV increased to 82.87 ± 3.29 fl compared with controls (78.73 ± 4.92 fl; p < 0.01). Conclusions: VPA therapy is associated with changes of the FA composition of membranes, which is not the case with PB therapy. The implications of this finding remain to be established. [ABSTRACT FROM AUTHOR]

Pyruvate decarboxylase (EC 4.1.1.1) was isolated and purified from the yeast Kluyveromyces lactis . The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments. The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K. lactis showed a minimum at a pyruvate concentration of 1.5 mm. According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first (regulatory) and the second (catalytic) substrate molecule. The microscopic rate constants of the substrate activation could be determined by a refined fit procedure. The influence of the artificial activator pyruvamide on the activation of the enzyme was studied. [ABSTRACT FROM AUTHOR]

Examines the crystal structure of alpha-carbanion in thiamin diphosphate. Reactions in transketolase reaction; Catalyst of ketol transfer between ketose and aldose sugar; Conformation of carbanion/enamine intermediate.

Unlabelled: Do children who remain in remission with epilepsy after withdrawal of anticonvulsive therapy still exhibit a different behaviour and performance-related abilities compared to healthy children? It was the aim of this study to investigate whether these children showed deviating psychiatric behaviour and/or neuropsychological function deficits as compared with a healthy control group and with children following occasional seizures which had never been treated. The study comprised 39 children with epilepsy after withdrawal of anticonvulsive therapy, 37 children with occasional seizures, and 39 matched healthy control children who were tested with regard to psychiatric and neuropsychological criteria. The patients were aged 6-15 years; 28 had a primarily generalised epilepsy, and 11 had a focal epilepsy. The patients remained free from seizures for more than 1 year. Psychiatric testing comprised the Child Behavior Checklist by Achenbach and Edelbrock and the Mannheim Parent Interview. Neuropsychological testing included non-verbal intelligence, visual perception, short-term memory and learning ability, arithmetics, movement coordination and motor skills, fine motor skills and verbal functions. After withdrawal from anticonvulsive therapy, the children with epilepsy did not exhibit any psychiatric disturbances, whereas those with occasional seizures frequently showed behaviour problems. After withdrawal from anticonvulsive therapy, no neuropsychological deficits were found in the children with epilepsy as compared to those with occasional seizures or to healthy children. Risk factors for poorer test results were first seizures occurring before the age of 3 years, delayed development, and neonatal problems.Conclusion: Children with epilepsy who remained without relapse after withdrawal from the anticonvulsive therapy did not show any psychiatric abnormalities and their performance was comparable to that of healthy control children, provided no risk factors were present. [ABSTRACT FROM AUTHOR]

Summary: Purpose: To estimate the incidence rate of epilepsies and epileptic syndromes in German children and adolescents aged 1 month to <15 years, and to provide data on their classification. Methods: A population-based prospective study was performed between July 1, 1999, and June 30, 2000. All children aged 1 month to <15 years with a newly diagnosed epilepsy or epileptic syndrome were recorded by private pediatricians, EEG laboratories, and the two University Children's Hospitals in the neighboring cities of Heidelberg and Mannheim. The diagnoses were classified according to the International Classification of Epilepsies and Epileptic Syndromes of the International League Against Epilepsy (ILAE). Results: The total age-adjusted annual incidence rate was 60/100,000 (95% confidence interval, 42–84), with the highest incidence in the first year of life (146/100,000). Focal epilepsies or epileptic syndromes (58%; incidence rate, 35/100,000) were more common than were generalized ones (39%; incidence rate, 24/100,000), and 3% (incidence rate, 2/100,000) of the epilepsies or epileptic syndromes were undetermined. The rate of idiopathic (47%; incidence rate, 29/100,000) and symptomatic or cryptogenic epilepsies (50%; incidence rate, 30/100,000) was equal. No significant difference in incidence between boys and girls was found. Conclusions: Incidence rates for epilepsy in German children aged 1 month to <15 years are about equal to those of other countries in Europe and North America. In accordance with studies from the United States and from many European countries, incidence was highest in the first year of life, and no difference in the incidence between girls and boys was found. In Germany as throughout Europe, idiopathic generalized epileptic syndromes are more often diagnosed than in the United States. [ABSTRACT FROM AUTHOR]

Focuses on a study which analyzes the structural basis of substrate activation in yeast pyruvate decarboxylase (PDC). Method used for the purification and crystallization of PDC from yeast strain; Use of molecular replacement for structure determination and refinement; Characteristics of the structure quality of the final model.

Examines the importance of enzymes in the autoregulation of yeast pyruvate decarboxylase (PdC) gene expression. Concept of Pdc autoregulation; Characterization of the mutant alleles Pdc1-8 and Pdc1-14; Expression of the mutant alleles in Escherichia coli.

To study the molecular structure and function of pyruvate decarboxylase (PDC) from plants the protein was isolated from pea seeds and partially characterised. The active enzyme which occurs in the form of higher oligomers consists of two different subunits appearing in SDS/PAGE and mass spectroscopy experiments. For further experiments, like X-ray crystallography. It was necessary to elucidate the protein sequence. Partial cDNA clones encoding pyruvate decarboxylase from seeds of Pisum sativum cv. Miko have been obtained by means of polymerase chain reaction techniques. The first sequence were found using degenerate oligonucleotide primers designated according to conserved amino acid sequences of known pyruvate decarboxylases. The missing parts of one cDNA were amplified applying the 3'- and 5' -rapid amplification of cDNA ends systems. The amino acid sequence deduced from the entire cDNA sequence displays strong similarity to pyruvate decarboxylases from other organisms, especially from plants. A molecular mass of 64 kDa was calculated for this protein correlating with estimations for the smaller subunit of the oligomeric enzymes. The PCR experiments led to at least three different clones representing the middle part of the PDC cDNA indicating the existence of three isozymes. Two of these isoforms could be confirmed on the protein level by sequencing tryptic peptides. Only anaerobically treated roots showed a positive signal for PDC mRNA in Northern analysis although the cDNA from imbibed seeds was successfully used for PCR. [ABSTRACT FROM AUTHOR]