Drug-resistant parasites and aggregated infection – early-season dynamics

We investigate the effect of spatial aggregation in the infection dynamics of nematode parasites in ruminants. We show that a high degree of spatial aggregation is likely to lead to a dramatically enhanced rate of invasion by drug-resistant strains. Received: 13 December 1999 / Revised version: 3 April 2000 / Published online: 4 October 2000     

New insights into viral structure and virus–cell interactions through proteomics

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus–cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV–cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.     

Myocardial bridging and endothelial dysfunction – Computational fluid dynamics study

Myocardial bridging (MB) is associated with endothelial dysfunction in patients with angina and non-obstructive coronary artery disease. This study aims to determine if there is a link between abnormal blood flow patterns and endothelial dysfunction in patients with MB. Ten patients with MB in their left anterior descending (LAD) artery were selected, 5 of whom had endothelial dysfunction and 5 had no endothelial dysfunction based on their response to acetylcholine. Similarly, 10 patients without MB in their LAD, 5 of whom had endothelial dysfunction and 5 of whom had no endothelial dysfunction, were studied as a control group. Transient computational fluid dynamics simulations were performed to derive wall shear stress (WSS) over the entire vessel including proximal, middle and distal segments. Patients with MB and endothelial dysfunction had lower WSS in the proximal LAD and greater WSS in the mid-LAD than patients with MB but without endothelial dysfunction. When comparing patients with endothelial dysfunction, those with MB had significantly lower shear stress in the proximal LAD (0.32 ± 0.14 Pa (with MB) vs 0.71 ± 0.38 Pa (without MB), p = 0.01) and greater shear stress in the mid-LAD (2.81 ± 1.20 Pa (with MB) vs 1.66 ± 0.31 Pa (without MB), p = 0.014) than patients without MB. Our findings demonstrated that the presence of MB significantly contributes to low WSS and endothelial dysfunction relationship.     

Beta-lactamase database (BLDB) – structure and function

Beta-Lactamase Database (BLDB) is a comprehensive, manually curated public resource providing up-to-date structural and functional information focused on this superfamily of enzymes with a great impact on antibiotic resistance. All the enzymes reported and characterised in the literature are presented according to the class (A, B, C and D), family and subfamily to which they belong. All three-dimensional structures of β-lactamases present in the Protein Data Bank are also shown. The characterisation of representative mutants and hydrolytic profiles (kinetics) completes the picture and altogether these four elements constitute the essential foundation for a better understanding of the structure-function relationship within this enzymes family. BLDB can be queried using different protein- and nucleotide-based BLAST searches, which represents a key feature of particular importance in the context of the surveillance of the evolution of the antibiotic resistance. BLDB is available online at http://bldb.eu without any registration and supports all modern browsers.     

Integrated MALDI-MS imaging and LC–MS techniques for visualizing spatiotemporal metabolomic dynamics in a rat stroke model

Spatiotemporal information about biomolecules is indispensable for precise pathological analysis, but it remains largely unclear. Here we show a novel analytical platform combing mass spectrometry imaging (MSI) with its complementary technique, liquid chromatography–mass spectrometry (LC–MS), to elucidate more comprehensive metabolic behaviors, with spatiotemporal information, in tissues. Analysis of a rat transient middle cerebral artery occlusion (MCAO) brain tissue after ischemia–reperfusion was performed to characterize the detailed metabolomic response to pathological alterations. To compare the spatially resolved metabolic state between ischemic and contralateral hemispheres of the MCAO brain, coronally sliced tissues were subjected to MSI. We also measured the metabolites extracted from three different cerebral regions, including whole cortex (CTX), hippocampus (HI) and corpus striatum (CPu), by LC–MS. In the ischemic hemisphere, significant metabolic changes at the CTX and CPu were observed after reperfusion, while not at the HI. A region-specific metabolic behavior was observed in amino acid and nucleotide metabolism, as well as in the TCA cycle. Correlation between MSI and LC–MS data was relatively high in the CTX and CPu. Combination of both MS platforms visualized the diverse spatiotemporal metabolic dynamics during pathological progress. Thus, our proposed strategy will contribute to the understanding of the complex pathogenesis of ischemia–reperfusion.     

Evolution and spatial structure interact to influence plant–herbivore population and community dynamics

An individual-based model of plant–herbivore interactions was developed to test the potentially interactive effects of explicit space and coevolution on population and community dynamics. Individual plants and herbivores resided in cells on a lattice and carried linked interaction genes. Interaction strength between individual plants and herbivores depended on concordance between these genes (gene-for-gene coevolution). Mating and dispersal among individuals were controlled spatially within variably sized neighbourhoods. Without evolution we observed high-frequency plant–herbivore oscillations (blue spectra) with small individual neighbourhoods, and stochastic fluctuations (white spectra) with large neighbourhoods. Evolution resulted in decreased interaction strength, decreased herbivore-induced plant mortality, increased population sizes, and longer-term fluctuations (reddened spectra). Small herbivore neighbourhoods led to herbivore extinction only with evolution. To explore the increased population size response to evolution we ran simulations without evolution while tuning plant–herbivore interaction strength from high to none. We found that herbivore populations were maximized at intermediate levels of interaction strength that coincided with the interaction strength achieved when the system tuned itself through evolution. Overall, our model shows that the small-scale details of phenotypically variable individual-level interactions, leading to evolutionary dynamics, affect large-scale population and community dynamics.     

Energetics and structure of alanine-rich α-helices via adaptive steered molecular dynamics

The energetics and hydrogen bonding profiles of the helix-to-coil transition were found to be an additive property and to increase linearly with chain length, respectively, in alanine-rich α-helical peptides. A model system of polyalanine repeats was used to establish this hypothesis for the energetic trends and hydrogen bonding profiles. Numerical measurements of a synthesized polypeptide Ac-Y(AEAAKA)_kF-NH₂ and a natural α-helical peptide a2N (1–17) provide evidence of the hypothesis’s generality. Adaptive steered molecular dynamics was employed to investigate the mechanical unfolding of all of these alanine-rich polypeptides. We found that the helix-to-coil transition is primarily dependent on the breaking of the intramolecular backbone hydrogen bonds and independent of specific side-chain interactions and chain length. The mechanical unfolding of the α-helical peptides results in a turnover mechanism in which a 3₁₀-helical structure forms during the unfolding, remaining at a near constant population and thereby maintaining additivity in the free energy. The intermediate partially unfolded structures exhibited polyproline II helical structure as previously seen by others. In summary, we found that the average force required to pull alanine-rich α-helical peptides in between the endpoints—namely the native structure and free coil—is nearly independent of the length or the specific primary structure.     

Science – or not? The status and dynamics of biotechnology

This review traces the emergence of biotechnology as a new scientific discipline since the 1980s, when it became a major economic force. Significant changes in theoretical perception, research strategies, aims, and experimental methods, mainly in genetic engineering techniques, occurred during this period. The article is based on an analysis of its scientific status over four decades: the 60s and 70s when work in the field proceeded in different disciplines with a low level of coherence and little integration, then a significant change during the 80s and 90s when common approaches and the merging of molecular biology and biochemical engineering created a new discipline. The analysis covers scientific highlights and outstanding technical progress, presenting two studies undertaken by scientific and governmental agencies in Germany and the USA, as well as results of interviews and a questionnaire dealing with the scientific status of biotechnology. Answers to the questionnaire were obtained from internationally known scientists and from young scientists with biotechnology degrees. The results collected trace the transition of biotechnology from heterogeneous specialties and approaches towards a scientific discipline of its own. A hypothesis is put forward suggesting a new common paradigm allowing for a coherent perception the of phenomena observed.     

Hidden hysteresis – population dynamics can obscure gene network dynamics

Background

Positive feedback is a common motif in gene regulatory networks. It can be used in synthetic networks as an amplifier to increase the level of gene expression, as well as a nonlinear module to create bistable gene networks that display hysteresis in response to a given stimulus. Using a synthetic positive feedback-based tetracycline sensor in E. coli, we show that the population dynamics of a cell culture has a profound effect on the observed hysteretic response of a population of cells with this synthetic gene circuit.

Results

The amount of observable hysteresis in a cell culture harboring the gene circuit depended on the initial concentration of cells within the culture. The magnitude of the hysteresis observed was inversely related to the dilution procedure used to inoculate the subcultures; the higher the dilution of the cell culture, lower was the observed hysteresis of that culture at steady state. Although the behavior of the gene circuit in individual cells did not change significantly in the different subcultures, the proportion of cells exhibiting high levels of steady-state gene expression did change.Although the interrelated kinetics of gene expression and cell growth are unpredictable at first sight, we were able to resolve the surprising dilution-dependent hysteresis as a result of two interrelated phenomena - the stochastic switching between the ON and OFF phenotypes that led to the cumulative failure of the gene circuit over time, and the nonlinear, logistic growth of the cell in the batch culture.

Conclusions

These findings reinforce the fact that population dynamics cannot be ignored in analyzing the dynamics of gene networks. Indeed population dynamics may play a significant role in the manifestation of bistability and hysteresis, and is an important consideration when designing synthetic gene circuits intended for long-term application.

     

The impact of high hydrostatic pressure on structure and dynamics of β-lactoglobulin

Methods

Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering experiments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of β-lactoglobulin (βLG) in aqueous solution.

Background

βLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dissociates to monomer below about pH 3.

Results

High-pressure structural results show that the dimer–monomer equilibrium, as well as the protein–protein interactions, are only slightly perturbed by pressure, and βLG unfolding is observed above a threshold value of 3000 bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the βLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity.

General significance

Results suggest that density and compressibility of water molecules in contact with the protein are key parameters to regulate the protein flexibility.     

Inhibition of HCV translation by disrupting the structure and interactions of the viral CRE and 3′ X-tail

A phylogenetically conserved RNA structure within the NS5B coding region of hepatitis C virus functions as a cis-replicating element (CRE). Integrity of this CRE, designated SL9266 (alternatively 5BSL3.2), is critical for genome replication. SL9266 forms the core of an extended pseudoknot, designated SL9266/PK, involving long distance RNA–RNA interactions between unpaired loops of SL9266 and distal regions of the genome. Previous studies demonstrated that SL9266/PK is dynamic, with ‘open’ and ‘closed’ conformations predicted to have distinct functions during virus replication. Using a combination of site-directed mutagenesis and locked nucleic acids (LNA) complementary to defined domains of SL9266 and its interacting regions, we have explored the influence of this structure on genome translation and replication. We demonstrate that LNAs which block formation of the closed conformation inhibit genome translation. Inhibition was at least partly independent of the initiation mechanism, whether driven by homologous or heterologous internal ribosome entry sites or from a capped message. Provision of SL9266/PK in trans relieved translational inhibition, and mutational analysis implied a mechanism in which the closed conformation recruits a cellular factor that would otherwise suppresses translation. We propose that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication.     

Understanding LuxS-based quorum sensing and its inhibition – molecular dynamics simulation study

Molecular dynamics simulations were successfully applied to LuxS protein and its protein–ligand complex using the newly developed force field parameters for the iron containing active site. To the best of our knowledge, this was the first attempt to develop force field parameters for the iron containing active site of the LuxS protein. From the simulations, catalytically important amino acid residues were identified which were found to stabilise the ligand. Two residues Glu57 and Asp77 were involved in polar interactions while the protein region in the range between amino acid residue 125 and 131 were predicted to facilitate the entry of ligand to the active site. Other residues like Arg65, Asp77, Ile78 and Ser79 were also recognised as ligand stabilising factors deduced from the simulation. These results were also found to be in good agreement with earlier studies and thus demonstrated the successful application of MD simulations to the LuxS protein. Moreover, the simulation data were expected to be considered for the development of rational approaches in order to identify new LuxS-based quorum sensing antagonists for the treatment of pathologies caused by resistant bacteria.     

Phytoplankton structure and dynamics in Lake Sanabria and Valparaíso reservoir (NW Spain)

The aim of this work is to compare the composition and seasonality of the phytoplankton population in a natural oligotrophic lake (Lake Sanabria) and a mesotrophic reservoir (Valparaíso). Both ecosystems are located on the Tera river course (NW Spain), which runs along an area of ancient metamorphic and plutonic rocks. Some physical and chemical parameters, chlorophyll a and phytoplankton biovolume were studied from monthly samples collected at different depths during the periods 1987–1989 (Lake Sanabria) and 1991–1992 (Valparaíso). Phytoplankton biovolume and chlorophyll a concentration were about five times higher in Valparaíso than in Lake Sanabria. Species composition (and main phytoplankton groups) were different. Valparaíso was highly dominated by diatoms and Lake Sanabria by cryptophytes and small chlorophytes. In spite of the fact that both sites were nitrogen limited, heterocystous cyanophytes (Anabaena sp.) were detected only in Valparaíso. The relationships between phytoplankton structure and trophic level, hydrological conditions and nitrate content are discussed.     

CODEHOP-mediated PCR – A powerful technique for the identification and characterization of viral genomes

Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) PCR primers derived from amino acid sequence motifs which are highly conserved between members of a protein family have proven to be highly effective in the identification and characterization of distantly related family members. Here, the use of the CODEHOP strategy to identify novel viruses and obtain sequence information for phylogenetic characterization, gene structure determination and genome analysis is reviewed. While this review describes techniques for the identification of members of the herpesvirus family of DNA viruses, the same methodology and approach is applicable to other virus families.     

Actin filament nucleation and elongation factors – structure–function relationships

The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tβ4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs–Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been “outsourced” to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.