Fundamental limits to position determination by concentration gradients

Position determination in biological systems is often achieved through protein concentration gradients. Measuring the local concentration of such a protein with a spatially varying distribution allows the measurement of position within the system. For these systems to work effectively, position determination must be robust to noise. Here, we calculate fundamental limits to the precision of position determination by concentration gradients due to unavoidable biochemical noise perturbing the gradients. We focus on gradient proteins with first-order reaction kinetics. Systems of this type have been experimentally characterised in both developmental and cell biology settings. For a single gradient we show that, through time-averaging, great precision potentially can be achieved even with very low protein copy numbers. As a second example, we investigate the ability of a system with oppositely directed gradients to find its centre. With this mechanism, positional precision close to the centre improves more slowly with increasing averaging time, and so longer averaging times or higher copy numbers are required for high precision. For both single and double gradients, we demonstrate the existence of optimal length scales for the gradients for which precision is maximized, as well as analyze how precision depends on the size of the concentration-measuring apparatus. These results provide fundamental constraints on the positional precision supplied by concentration gradients in various contexts, including both in developmental biology and also within a single cell.     

On the use of oxygenic photosynthesis for the sustainable production of commodity chemicals

A sustainable society will have to largely refrain from the use of fossil carbon deposits. In such a regime, renewable electricity can be harvested as a primary source of energy. However, as for the synthesis of carbon‐based materials from bulk chemicals, an alternative is required. A sustainable approach towards this is the synthesis of commodity chemicals from CO₂, water and sunlight. Multiple paths to achieve this have been designed and tested in the domains of chemistry and biology. In the latter, the use of both chemotrophic and phototrophic organisms has been advocated. ‘Direct conversion’ of CO₂ and H₂O, catalyzed by an oxyphototroph, has excellent prospects to become the most economically competitive of these transformations, because of the relative ease of scale‐up of this process. Significantly, for a wide range of energy and commodity products, a proof of principle via engineering of the corresponding production organism has been provided. In the optimization of a cyanobacterial production organism, a wide range of aspects has to be addressed. Of these, here we will put our focus on: (1) optimizing the (carbon) flux to the desired product; (2) increasing the genetic stability of the producing organism and (3) maximizing its energy conversion efficiency. Significant advances have been made on all these three aspects during the past 2 years and these will be discussed: (1) increasing the carbon partitioning to >50%; (2) aligning product formation with the growth of the cells and (3) expanding the photosynthetically active radiation region for oxygenic photosynthesis.     

Deamidations in recombinant human phenylalanine hydroxylase. Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.     

Comparing approximations to spatio-temporal models for epidemics with local spread

Analytical methods for predicting and exploring the dynamics of stochastic, spatially interacting populations have proven to have useful application in epidemiology and ecology. An important development has been the increasing interest in spatially explicit models, which require more advanced analytical techniques than the usual mean-field or mass-action approaches. The general principle is the derivation of differential equations describing the evolution of the expected population size and other statistics. As a result of spatial interactions no closed set of equations is obtained. Nevertheless, approximate solutions are possible using closure relations for truncation. Here we review and report recent progress on closure approximations applicable to lattice models with nearest-neighbour interactions, including cluster approximations and elaborations on the pair (or pairwise) approximation. This study is made in the context of an SIS model for plant-disease epidemics introduced in Filipe and Gibson (1998, Studying and approximating spatio-temporal models for epidemic spread and control, Phil. Trans. R. Soc. Lond. B 353, 2153–2162) of which the contact process [Harris, T. E. (1974), Contact interactions on a lattice, Ann. Prob. 2, 969] is a special case. The various methods of approximation are derived and explained and their predictions are compared and tested against simulation. The merits and limitations of the various approximations are discussed. A hybrid pairwise approximation is shown to provide the best predictions of transient and long-term, stationary behaviour over the whole parameter range of the model.     

Spatial and temporal organization of replicating Escherichia coli chromosomes

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.     

Reference Ranges for Uterine Artery Pulsatility Index during the Menstrual Cycle: A Cross-Sectional Study

Background

Cyclic endometrial neoangiogenesis contributes to changes in local vascular patterns and is amenable to non-invasive assessment with Doppler sonography. We hypothesize that the uterine artery (UtA) impedance, measured by its pulsatility index (PI), exhibits a regular pattern during the normal menstrual cycle. Therefore, the main study objective was to derive normative new day-cycle-based reference ranges for the UtA-PI during the entire cycle from days 1 to 34 according to the isolated time effect and potential confounders such as age and parity.

Methods

From January 2009 to December 2012, a cross-sectional study of 1,821 healthy women undergoing routine gynaecological ultrasound was performed. The Doppler flow of the right and left UtA-PI was studied transvaginally by colour and pulsed Doppler imaging. The mean right and left values and the presence or absence of a bilateral protodiastolic notch were recorded. Reference intervals for the PI according to the cycle day were generated by classical linear regression.

Results

The majority of patients (97.5%) presented unilateral or bilateral UtA notches. The crude 5th, 50th, and 95th reference percentile curves of the UtA-PI at 1–34 days of the normal menstrual cycle were derived. In all curves, a progressive significant decrease occurred during the first 13 days, followed by an increase and recovery in the UtA-PI. The adjusted 5th, 50th, and 95th reference percentile curves for the effects of age and parity were also obtained. These two conditions generated an approximately identical UtA-PI pattern during the cycle, except with small but significant reductions at the temporal extremes.

Conclusions

The median, 5th, and the 95th percentiles of the UtA-PI decrease during the first third of the menstrual cycle and recover to their initial values during the last two thirds of the cycle. The rates of decrease and recovery depend significantly on age and parity.     

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.