Position Effect Takes Precedence Over Target Sequence in Determination of Adenine Methylation Patterns in the Nuclear Genome of a Eukaryote, \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5′-NAT-3′, but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation

Oral lorazepam can be substituted for intravenous midazolam when weaning paediatric intensive care patients off sedation

Aim: Intravenous sedatives used in the paediatric intensive care unit (PICU) need to be tapered after prolonged use to prevent ia

Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes – with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes. By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92 mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli. The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes

Development of enzymatic activity during protein folding. Detection of a spectroscopically silent native-like intermediate of muscle acylphosphatase.

The recovery of enzymatic activity during the folding of muscle acylphosphatase and two single residue mutants (proline 54 to alanine and proline 71 to alanine) from 7 M urea has been monitored and compared with the development of intrinsic fluorescence emission. Fluorescence measurements reveal the presence in the wild-type protein of a major rapid refolding phase followed by a second low amplitude slow phase. The slow phase is absent in the fluorescence trace acquired with the proline 54 to alanine mutant, suggesting the involvement of this proline residue in the fluorescence-detected slow phase of the wild-type protein. The major kinetic phase is associated with a considerable recovery of enzymatic activity, indicating that a large fraction of molecules refolds with effective two-state behavior. The use of time-resolved enzymatic activity as a probe to follow the folding process reveals, however, the presence of another exponential slow phase arising from proline 71. This slow phase is not observable by utilizing optical probes, indicating that, unlike proline 54, the cis to trans isomerization of proline 71 can take place in an intermediate possessing a native-like fold. We suggest that, although spectroscopically silent and structurally insignificant, the cis-trans interconversion of proline residues in native-like intermediates may be crucial for the generation of enzymatic activity of functional enzymes

Mycorrhizal mycelium as a global carbon pool

For more than 400 million years, mycorrhizal fungi and plants have formed partnerships that are crucial to the emergence and functioning of global ecosystems. The importance of these symbiotic fungi for plant nutrition is well established. However, the role of mycorrhizal fungi in transporting carbon into soil systems on a global scale remains under-explored. This is surprising given that ∼75% of terrestrial carbon is stored belowground and mycorrhizal fungi are stationed at a key entry point of carbon into soil food webs. Here, we analyze nearly 200 datasets to provide the first global quantitative estimates of carbon allocation from plants to the mycelium of mycorrhizal fungi. We estimate that global plant communities allocate 3.93 Gt CO2e per year to arbuscular mycorrhizal fungi, 9.07 Gt CO2e per year to ectomycorrhizal fungi, and 0.12 Gt CO2e per year to ericoid mycorrhizal fungi. Based on this estimate, 13.12 Gt of CO2e fixed by terrestrial plants is, at least temporarily, allocated to the underground mycelium of mycorrhizal fungi per year, equating to ∼36% of current annual CO2 emissions from fossil fuels. We explore the mechanisms by which mycorrhizal fungi affect soil carbon pools and identify approaches to increase our understanding of global carbon fluxes via plant–fungal pathways. Our estimates, although based on the best available evidence, are imperfect and should be interpreted with caution. Nonetheless, our estimations are conservative, and we argue that this work confirms the significant contribution made by mycorrhizal associations to global carbon dynamics. Our findings should motivate their inclusion both within global climate and carbon cycling models, and within conservation policy and practice

Identifying context factors explaining physician's low performance in communication assessment: an explorative study in general practice

Contains fulltext : 97982.pdf (postprint version ) (Open Access)ABSTRACT: BACKGROUND: Communication is a key competence for health care professionals. Analysis of registrar and GP communication performance in daily practice, however, suggests a suboptimal application of communication skills. The influence of context factors could reveal why communication performance levels, on average, do not appear adequate. The context of daily practice may require different skills or specific ways of handling these skills, whereas communication skills are mostly treated as generic. So far no empirical analysis of the context has been made. Our aim was to identify context factors that could be related to GP communication. METHODS: A purposive sample of real-life videotaped GP consultations was analyzed (N = 17). As a frame of reference we chose the MAAS-Global, a widely used assessment instrument for medical communication. By inductive reasoning, we analyzed the GP behaviour in the consultation leading to poor item scores on the MAAS-Global. In these cases we looked for the presence of an intervening context factor, and how this might explain the actual GP communication behaviour. RESULTS: We reached saturation after having viewed 17 consultations. We identified 19 context factors that could potentially explain the deviation from generic recommendations on communication skills. These context factors can be categorized into doctor-related, patient-related, and consultation-related factors. CONCLUSIONS: Several context factors seem to influence doctor-patient communication, requiring the GP to apply communication skills differently from recommendations on communication. From this study we conclude that there is a need to explicitly account for context factors in the assessment of GP (and GP registrar) communication performance. The next step is to validate our findings

Robust structure-based resonance assignment for functional protein studies by NMR

High-throughput functional protein NMR studies, like protein interactions or dynamics, require an automated approach for the assignment of the protein backbone. With the availability of a growing number of protein 3D structures, a new class of automated approaches, called structure-based assignment, has been developed quite recently. Structure-based approaches use primarily NMR input data that are not based on J-coupling and for which connections between residues are not limited by through bonds magnetization transfer efficiency. We present here a robust structure-based assignment approach using mainly HN–HN NOEs networks, as well as 1H–15N residual dipolar couplings and chemical shifts. The NOEnet complete search algorithm is robust against assignment errors, even for sparse input data. Instead of a unique and partly erroneous assignment solution, an optimal assignment ensemble with an accuracy equal or near to 100% is given by NOEnet. We show that even low precision assignment ensembles give enough information for functional studies, like modeling of protein-complexes. Finally, the combination of NOEnet with a low number of ambiguous J-coupling sequential connectivities yields a high precision assignment ensemble. NOEnet will be available under: http://www.icsn.cnrs-gif.fr/download/nmr

A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms

Assessing fitness-to-practice of overseas-trained health practitioners by Australian registration & accreditation bodies

Assessment of fitness-to-practice of health professionals trained overseas and who wish to practice in Australia is undertaken by a range of organisations. These organisations conduct assessments using a range of methods. However there is very little published about how these organisations conduct their assessments. The purpose of the current paper is to investigate the methods of assessment used by these organisations and the issues associated with conducting these assessments

Testing the hierarchical SDT model: the case of performance-oriented classrooms

Testing the hierarchical SDT model: the case of performance-oriented classrooms