Pattern of accessory regions and invasive disease potential in Streptococcus pneumoniae.

BACKGROUND: The invasive disease potential (IDP) of Streptococcus pneumoniae differs between serotypes, but the reason for this is unknown. METHODS: A total of 47 pneumococcal isolates from 13 serotypes with different IDPs in humans that belonged to 37 multilocus sequence types were compared by whole genome microarrays and mutant analyses. RESULTS: Approximately 34% of the genes were variable, including 95 genes previously shown by signature-tagged mutagenesis (STM) to be required for invasive disease in mice. Many variable genes were localized to 41 accessory regions (ARs), of which 24 contained genes previously identified by STM as required for invasive disease. Only AR6 and AR34 were preferentially found in isolates of serotypes with high IDPs. Neither AR6, which carries a gene previously identified bySTMas required for invasive disease and encodes a 6-phospho-betaglucosidase, nor the putative adhesin expressed by AR34 was required for mouse virulence in TIGR4. CONCLUSIONS: Pneumococci possess a repertoire of ARs that differ between clones and even between isolates of the same clone. The ARs required for invasive disease in humans may be redundant, as no unique pattern distinguished the most invasive clones from others. The ARs that contained genes previously identified by STM as required for virulence in mice were frequently absent from invasive human isolates. Only 1 AR (AR6) was present in almost all isolates from the serotypes with the highest IDP (1, 4, and 7F), whereas it was missing from many others.

Clonal success of piliated penicillin nonsusceptible pneumococci.

Antibiotic resistance in pneumococci is due to the spread of strains belonging to a limited number of clones. The Spain(9V)-3 clone of sequence type (ST)156 is one of the most successful clones with reduced susceptibility to penicillin [pneumococci nonsusceptible to penicillin (PNSP)]. In Sweden during 2000-2003, a dramatic increase in the number of PNSP isolates was observed. Molecular characterization of these isolates showed that a single clone of sequence type ST156 increased from 40% to 80% of all serotype 14, thus causing the serotype expansion. Additionally, during the same time period, we examined the clonal composition of two serotypes 9V and 19F: all 9V and 20% of 19F isolates belonged to the clonal cluster of ST156, and overall approximately 50% of all PNSP belonged to the ST156 clonal cluster. Moreover, microarray and PCR analysis showed that all ST156 isolates, irrespective of capsular type, carried the rlrA pilus islet. This islet was also found to be present in the penicillin-sensitive ST162 clone, which is believed to be the drug-susceptible ancestor of ST156. Competitive experiments between related ST156 serotype 19F strains confirmed that those containing the rlrA pilus islet were more successful in an animal model of carriage. We conclude that the pilus island is an important biological factor common to ST156 isolates and other successful PNSP clones. In Sweden, a country where the low antibiotic usage does not explain the spread of resistant strains, at least 70% of all PNSP isolates collected during year 2003 carried the pilus islet.

Channel density regulation of firing patterns in a cortical neuron model.

Modifying the density and distribution of ion channels in a neuron (by natural up- and downregulation or by pharmacological intervention or by spontaneous mutations) changes its activity pattern. In this investigation we analyzed how the impulse patterns are regulated by the density of voltage-gated channels in a neuron model based on voltage-clamp measurements of hippocampal interneurons. At least three distinct oscillatory patterns, associated with three distinct regions in the Na-K channel density plane, were found. A stability analysis showed that the different regions are characterized by saddle-node, double-orbit, and Hopf-bifurcation threshold dynamics, respectively. Single, strongly graded action potentials occur in an area outside the oscillatory regions, but less graded action potentials occur together with repetitive firing over a considerable range of channel densities. The relationship found here between channel densities and oscillatory behavior may partly explain the difference between the principal spiking patterns previously described for crab axons (class 1 and 2) and cortical neurons (regular firing and fast spiking).

Na channel kinetics: developing models from non-stationary current fluctuations by analytic methods.

In a previous study, we analyzed Na current fluctuations in myelinated axons from Xenopus laevis under voltage clamp conditions. The statistical properties were analyzed in terms of covariance functions for consecutive time intervals of varying duration during the pulse step. The underlying channel kinetics was analyzed by performing stochastic simulations of published Na channel models and calculating corresponding covariance functions. None of the models explained the fluctuation results. We therefore developed a novel minimal Na channel model that satisfactorily described the results. In the present paper, we extend the analysis and specify the possible models explaining the experimental data by using analytical methods. We derive general relations between the experimental data, including the covariance functions, and the rate constants of specific one-open-state models. A general feature of these models is that they comprise an inactivation step from the first closed state and a relatively low backward rate from the open state. This is in accordance the minimal model inferred from numerical stochastic calculations in the previous study.

On the appearance of function and organisation in the origin of life.

Models for the steps of organisation in the origin of life are discussed with an emphasis on stability, and the possibilities of acquiring a diversity of functions. In particular, two basic models are described: that of simple self-replicating molecules, and that of autocatalytic self-reproduction, which is accomplished by a hypercyclic organisation. The latter may be exemplified by the RNA world. The view of a step-wise development with new functions successively incorporated and a high accuracy of the reproduction from the onset is criticised. Instead, we suggest that no clear systematic information is continued to the first cell before the start of protein synthesis. A non-selective manifold of self-replicating molecules and unsystematic protein production from the beginning could have caused a very large diversity from which functions that could stabilise the system by feedback loops could be selected. The only way to stabilise the protein synthesis and the genetic code would be to have feedback mechanisms so that the code actually produced the proteins that supported that very code. The code would then become frozen. As DNA would require control functions, it would not be used as a single information-carrier until protein synthesis had been established and the functions were available.

Logics and logistics of community intervention against osteoporosis: an evidence basis.

Under designations like small areas action research and intervention, directed 'ground-up' health promotion and prevention in the population form an important part of the ongoing medical systems development. There is recent evidence of the success of community intervention against cardiovascular disease. In osteoporosis, however, there is still a lack of conclusive data on both the logics and logistics of such an approach. Since 1988, a county health policy program has been formulated and implemented in Ostergötland, Sweden, following the principles and guidelines of the WHO HFA 2000 declaration. Vadstena (n approximately 7,600) was chosen for a local and generalizable osteoporosis prevention project mediated by the primary care organization by means of health promotion and education in the community. In the present report we emphasize that community intervention is an important new advancement of the medical systems, where the basic research questions include operational and management aspects as equally vital and measurable requisites and results as other performance and outcome variables. We found that a community intervention trial against osteoporosis is both motivated and feasible and in this report wish to provide evidence on these crucial issues of logics and logistics.

The Professional Indemnity Review: what did it accomplish?

In a previous article,* Richard Tjiong criticised the Professional Indemnity Review's specific recommendations for reform of professional indemnity insurance. But the Review covered many other issues, particularly to do with identifying, evaluating and reducing adverse outcomes of medical procedures. Charlotta Blomberg highlights some of the key findings (and failings) of the Review's Final Report.

The role of accuracy for early stages of the origin of life.

The role of accuracy in developing stages in the origin of life is investigated by mathematical models. Errors yield difficult stability problems in a description of a cooperative system with information-carriers (genes) and catalytic polymers (enzymes) that are related to each other. This problem is highly relevant for a proper understanding of the RNA world. The model that is treated takes two relevant error effects, which can be coupled, into account. One is the occurrence of parasites, inactive genes that are efficiently reproduced by the catalytic support of other polymers but which do not give rise to any catalytic activity. The other is an error propagation due to the fact that erroneous catalysts will provide an increased error level and thus more erroneous catalysts which may lead to still increasing error levels, eventually an error catastrophe.

A model of error propagation in the presence of an error-enhancing drug.

A mathematical model is considered that describes error propagation in protein synthesis, with emphasis on the role of an error-enhancing drug such as streptomycin. The subject of error propagation has been investigated in a number of works since an original proposal by Orgel in 1963. From experiments, it is known that streptomycin given to a bacterium culture in small concentration increases the error level, but at a certain concentration threshold the bacteria die. This two-fold behavior has, in some papers, lead to the proposal that another effect of the drugs besides that of error propagation leads to the death of the bacteria. In the present work, we use a model related to kinetic models of selection in protein synthesis which include the combined effect of ribosome and synthetase action. The model shows very clear threshold effects: for small values of the parameter that represents the drug action, a stable situation is found that has an increased error level but still attains a rather high accuracy. At a certain threshold, this is no longer maintained. The main emphasis here is on the time evolution of relevant parameters, and it is also shown how this can be quite drastic: the accuracy may decrease rather smoothly to a critical point, where it is drastically lost and where the bacteria may die out very suddenly.

Predictive testing for multiple endocrine neoplasia type 1 using DNA polymorphisms.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominantly inherited predisposition to neoplastic lesions of the parathyroids, pancreas, and the pituitary. We have previously located the predisposing genetic defect to the long arm of chromosome 11 by genetic linkage. In this study, 124 members of six MEN1 families, including 59 affected individuals, were genotyped for restriction fragment length polymorphisms with different DNA probes, and the genetic linkage between these marker systems and MEN1 was determined. 13 marker systems (17 DNA probes) were found to be linked to MEN1. These markers are located within a region on chromosome 11 spanning 14% meiotic recombinations, with the MEN1 locus in the middle. Four of the marker systems are on the centromeric side of MEN1, and four on the telomeric side, based on meiotic crossovers. The remaining five DNA probes are closely linked to MEN1, with no crossovers in our set of families. The 13 marker systems can be used for an accurate and reliable premorbid test for MEN1. In most clinical situations it is possible to identify a haplotype of this part of chromosome 11 with the mutant MEN1 allele in the middle. The calculated predictive accuracy is greater than 99.5% if three such marker systems are informative. Therefore, genetic linkage testing can be used for informed genetic counseling in MEN1 families, and to avoid unnecessary biochemical screening programs.

Localization of the MEN1 gene to a small region within chromosome 11q13 by deletion mapping in tumors.

The gene for multiple endocrine neoplasia type 1 (MEN1), an inherited predisposition to neuroendocrine neoplasm of the parathyroid glands, the pancreatic islet parenchyma, and the anterior pituitary gland, was recently mapped to chromosome 11q13 based on genetic linkage in families. We now show that the pathogenesis of MEN1-associated parathyroid lesions involves unmasking of a recessive mutation at the disease locus and that sporadic primary hyperparathyroidism shares the same mechanisms. By examination of allele losses in MEN1-associated lesions, we could define deletions of chromosome 11 and map the MEN1 locus to a small region within chromosome band 11q13, telomeric to the PYGM locus. In contrast, a low incidence of deletions involving the MEN1 gene was found in sporadic pituitary adenomas.

Modelling efficiency, error propagation and the effect of error-enhancing drugs in protein synthesis.

The efficiency of protein synthesis is discussed with the main emphasis on the accuracy. The error increase by erroneous synthesizing proteins is studied by a theoretical model which includes the coupling of the amino acylation step to the protein synthesis at the ribosomes. Depending on parameter values, the model yields a stable point of relatively high accuracy, a point where most of the accuracy is lost and virtually no functioning proteins are produced (error catastrophe), or both. The effect of an error enhancing drug such as streptomycin can be considered, and it is found that one can have a situation where small amounts of the drug yields a decreased but stable accuracy, and where this possibility disappears at a certain drug level. At higher drug levels, the cells cannot attain any accuracy. The transition is quite drastic, and analogous to a phase transition.

Site dependent time optimization of protein synthesis with special regard to accuracy.

The efficiency of protein synthesis is determined by its rate, accuracy, and energy consumption. With the energy consumption fixed, we optimize the system with respect to time and accuracy. Using an analytic model for a simple system and computer simulations for more complex systems, where also the possibility of errors is included, we demonstrate how different parts of the messenger RNA influence the protein production rate differently. The first part of the coding sequence is of major importance, since the availability of empty initiation sites is crucial, and queuing back to that region may interfere with initiation. The elongation rate at different positions depends on codon usage, on the concentrations of substrate and co-factors, and on the kinetic rate constants, including those of the proofreading branch(es). Ribosomal proofreading is a time consuming process and by allowing for more errors in the beginning of a protein, it is possible to increase the production rate of that protein. We calculate the mean translation time per functioning protein for various translation accuracies, and discuss the different strategies open to living cells.

Free energy and time economy for the mutual selection of monomers in biosynthesis, primarily protein synthesis.

The starting point of this work is the fact that the correction of errors in biosynthesis must be paid for by an increased dissipation (free energy loss) or a time delay. Further, a low accuracy is wasteful in this respect as the cell then produces a number of non-functioning products with a significant "production cost". One can then look for the situations of best "economy" for the selection processes. This is particularly obvious in reciprocal selections, where in some cases a substrate A shall be selected but discriminated against a competitor B, and in other cases, the opposite is true, B shall be selected with A as a competitor. It can be expected only in certain symmetric situations that these reciprocal selections are made in an equal way. Because one substrate shall be selected more often or it may be more relevant for the product, it may occur in higher concentrations and/or be selected more accurately (at a higher cost). The opposite selection may then be less accurate. The work studies various aspects of this.

Theoretical modelling of protein synthesis.

This article provides an overview of the use of mathematical and computer modelling in furthering the understanding of protein synthesis. In particular, we discuss issues such as the nature of the rate limiting step(s), error rates, tRNA-codon adaptation, codon bias, attenuation control, and problems of selection and error corrections, focussing on their theoretical treatment.

Optimization of error-correction processes with respect to time. Comparison to free energy aspects.

Time aspects of selection processes with the possibility of error correction through proofreading branches are studied by mathematical modelling of the kinetics of the reaction network. The methods are similar to those previously developed for free energy aspects. The minimum time delay that is necessary for achieving a certain accuracy level can then be calculated. The main difference to previous results lies in the initial association-dissociation step. In the free energy picture, this shall be essentially equilibrated, but that would yield a too large time delay in the time picture. Characteristic features that are indicative for the optimization strategy of the cell are discussed.

Error propagation in E. coli protein synthesis.

A new approach to the error catastrophe theory, proposed by Leslie Orgel, is presented here. Our model is a development of previous models, but differs in several respects: the overall activity is assumed to be dependent on the error level, the effect of errors in the translating system, giving rise to additional errors in the succeeding generation of products, is explicitly included as a special term in our model, and scavenging enzymes are assumed to break down and eliminate products with a loose structure. Their efficiency is dependent on the error level. The model also takes into account the dilution of incorrect ribosomes and enzymes, and is described by a time-dependence in terms of ribosome/enzyme generations. The model and the contribution to the time development are discussed in the light of experiments on E. coli treated with streptomycin.

The tRNA cycle and its relation to the rate of protein synthesis.

With the aid of a kinetic model, we have investigated how the adaptation between the various components of the tRNA cycle and the codon frequencies affects the rate of protein synthesis. Depending on the relative amounts of total tRNA, synthetase and ribosomes, the optimal correlations vary between a situation where all tRNA species are either present in equal amounts or are present in amounts proportional to the square-root of the corresponding codon frequencies, and a situation where the amounts of the different tRNA species present are linearly proportional to the codon frequencies.