A novel nucleotide implicated in the response of E. coli to energy source downshift

When E. coli cells are subjected to energy source downshift, the accumulation of RNA (and overall cell growth) is drastically restricted within 1 to 2 min. However, the identity of the primary metabolic signal for this adjustment is a mystery. Earlier studies, and further evidence presented here, show that there is no satisfactory correlation between the sudden adjustment of RNA accumulation and the kinetics of changes in the levels of prospective signalling compounds, such as glycolytic intermediates, ppGpp, ATP, or the three adenylate nucleotides. We have discovered an unusual nucleotide, which we call the phantom spot, whose level decreases dramatically within a minute of downshift, correlating well with the adjustment of RNA accumulation. Preliminary characterization of the phantom spot indicates that it is a triphosphate derived from the guanylate pathway, and suggests that it is a form of GTP with a modification of the imidazole portion of the purine ring. We postulate that this nucleotide serves as a regulatory facsimile of ATP, linking the rate of RNA accumulation and other anabolic processes to the overall rate of phosphorylation.     

Research in general practice: a survey of incentives and disincentives for research participation

Background Recruitment rates of general practitioners (GPs) to do research vary widely. This may be related to the ability of a study to incorporate incentives for GPs and minimise barriers to participation.Method A convenience sample of 30 GPs, ten each from the Sydney intervention and control groups Ageing in General Practice ‘Detection and Management of Dementia’ project (GP project) and 10 GPs who had refused participation, were recruited to determine incentives and barriers to participating in research. GPs completed the 11-item ‘Meeting the challenges of research in general practice: general practitioner questionnaire’ (GP survey) between months 15 and 24 of the GP project, and received brief qualitative interviews from a research GP to clarify responses where possible.Results The most important incentives the 30 GPs gave for participating in the project were a desire to update knowledge (endorsed by 70%), to help patients (70%), and altruism (60%). Lack of time (43%) was the main barrier. GPs also commented on excessive paperwork and an inadequate explanation of research.Conclusions While a desire to update knowledge and help patients as well as altruism were incentives, time burden was the primary barrier and was likely related to extensive paperwork. Future recruitment may be improved by minimising time burden, making studies simpler with online data entry, offering remuneration and using a GP recruiter.     

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (¹O₂) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect ¹O₂ phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of ¹O₂ phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with ¹O₂ phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time ¹O₂ dosimetry during PDT treatment using CW light.     

Predicting Peptide Structures in Native Proteins from Physical Simulations of Fragments

It has long been proposed that much of the information encoding how a protein folds is contained locally in the peptide chain. Here we present a large-scale simulation study designed to examine the extent to which conformations of peptide fragments in water predict native conformations in proteins. We perform replica exchange molecular dynamics (REMD) simulations of 872 8-mer, 12-mer, and 16-mer peptide fragments from 13 proteins using the AMBER 96 force field and the OBC implicit solvent model. To analyze the simulations, we compute various contact-based metrics, such as contact probability, and then apply Bayesian classifier methods to infer which metastable contacts are likely to be native vs. non-native. We find that a simple measure, the observed contact probability, is largely more predictive of a peptide''s native structure in the protein than combinations of metrics or multi-body components. Our best classification model is a logistic regression model that can achieve up to 63% correct classifications for 8-mers, 71% for 12-mers, and 76% for 16-mers. We validate these results on fragments of a protein outside our training set. We conclude that local structure provides information to solve some but not all of the conformational search problem. These results help improve our understanding of folding mechanisms, and have implications for improving physics-based conformational sampling and structure prediction using all-atom molecular simulations.     

SplitsTree: analyzing and visualizing evolutionary data

MOTIVATION: Real evolutionary data often contain a number of different and sometimes conflicting phylogenetic signals, and thus do not always clearly support a unique tree. To address this problem, Bandelt and Dress (Adv. Math., 92, 47-05, 1992) developed the method of split decomposition. For ideal data, this method gives rise to a tree, whereas less ideal data are represented by a tree-like network that may indicate evidence for different and conflicting phylogenies. RESULTS: SplitsTree is an interactive program, for analyzing and visualizing evolutionary data, that implements this approach. It also supports a number of distances transformations, the computation of parsimony splits, spectral analysis and bootstrapping.      

Effect of calcium ions on creatine kinase systems of myocardial cells

The kinetics of the creatine phosphokinase reaction catalyzed by different creatine phosphokinase (CPK) isoenzymes from the heart and the mechanism of the regulatory action of calcium ions on this reaction have been studied. It has been shown that the kinetic parameters of the reaction in the presence of calcium are similar to those in the presence of magnesium with the exception of the maximal rate of the reverse reaction, which is about three times lower in the presence of calcium. Calcium ions are able to exert a significant regulatory action on the CPK reaction. Simultaneous regulation of the CPK reaction by calcium and magnesium ions can be quantitatively described by a kinetic model, which takes into account the formation of complexes of adenine nucleotides with calcium and magnesium. The mechanism of the CPK reaction regulation by metal ions involves changes in concentrations of the metal -- adenine nucleotide compexes. The regulatory action of calcium on the creatine kinase reaction is the same for all CPK isoenzymes studied.