Effect of membrane stiffness and cytoskeletal element density on mechanical stimuli within cells: an analysis of the consequences of ageing in cells

A finite element model of a single cell was created and used to investigate the effects of ageing on biophysical stimuli generated within a cell. Major cellular components were incorporated in the model: the membrane, cytoplasm, nucleus, microtubules, actin filaments, intermediate filaments, nuclear lamina, and chromatin. The model used multiple sets of tensegrity structures. Viscoelastic properties were assigned to the continuum components. To corroborate the model, a simulation of Atomic Force Microscopy (AFM) indentation was performed and results showed a force/indentation simulation with the range of experimental results.

Ageing was simulated by both increasing membrane stiffness (thereby modelling membrane peroxidation with age) and decreasing density of cytoskeletal elements (thereby modelling reduced actin density with age). Comparing normal and aged cells under indentation predicts that aged cells have a lower membrane area subjected to high strain compared to young cells, but the difference, surprisingly, is very small and would not be measurable experimentally. Ageing is predicted to have more significant effect on strain deep in the nucleus. These results show that computation of biophysical stimuli within cells are achievable with single-cell computational models whose force/displacement behaviour is within experimentally observed ranges. the models suggest only small, though possibly physiologically-significant, differences in internal biophysical stimuli between normal and aged cells.

A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress

Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB-TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB-TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H(+) antiport.

Neuromusculature of Gyrodactylus rysavyi, a monogenean gill and skin parasite of the catfish Clarias gariepinus

Phalloidin fluorescence technique, enzyme cytochemistry and immunocytochemistry in conjunction with confocal scanning laser microscopy were used for the first time to describe the nervous and muscle systems of the viviparous monogenean parasite, Gyrodactylus rysavyi inhabiting the gills and skin of the Nile catfish Clarias gariepinus. The body wall muscles are composed of an outer layer of circular fibres, an intermediate layer of paired longitudinal fibres and an inner layer of well-spaced bands of diagonal fibres arranged in two crossed directions. The musculature of the pharynx, intestine, reproductive tract and the most prominent muscles of the haptor were also described. Two characteristic muscular pads were found lying in the anterior region of the haptor in close contact with the hamuli. To each one of these pads, a group of ventral extrinsic muscles was connected. The role of this ventral extrinsic muscle in the body movement was discussed. The mechanism operating the marginal hooklets was also discussed. The central nervous system (CNS) consists of paired cerebral ganglia from which three pairs of longitudinal ventral, lateral and dorsal nerve cords arise. The nerve cords are connected at intervals by many transverse connectives. The CNS is better developed ventrally than dorsally or laterally and it has the highest reactivity for all neuroactive substances examined. Both the central and the peripheral nervous system (PNS) are bilaterally symmetrical. Structural and functional correlates of the neuromusculature of the pharynx, haptor and reproductive tracts were explained. The results implicated acetylcholine, FMRFamide-related peptides (FaRPs) and serotonin in sensory and motor function. The results were compared with those of the monogeneans Macrogyrodactylus clarii and M. congolensis inhabiting the gills and skin respectively of the same host fish C. gariepinus.

Spitzer observations of the asteroid-comet transition object and potential spacecraft target 107P (4015) Wilson-Harrington

Context: Near-Earth asteroid-comet transition object 107P/ (4015) Wilson-Harrington is a possible target of the joint European Space Agency (ESA) and Japanese Aerospace Exploration Agency (JAXA) Marco Polo sample return mission. Physical studies of this object are relevant to this mission, and also to understanding its asteroidal or cometary nature. Aims: Our aim is to obtain significant new constraints on the surface thermal properties of this object. Methods: We present mid-infrared photometry in two filters (16 and 22 μm) obtained with NASA's Spitzer Space Telescope on February 12, 2007, and results from the application of the Near Earth Asteroid Thermal Model (NEATM). We obtained high S/N in two mid-IR bands allowing accurate measurements of its thermal emission. Results: We obtain a well constrained beaming parameter (η = 1.39±0.26) and obtain a diameter and geometric albedo of D = 3.46±0.32 km, and pV = 0.059±0.011. We also obtain similar results when we apply this best-fitting thermal model to single-band mid-IR photometry reported by Campins et al. (1995, P&SS, 43, 733), Kraemer et al. (2005, AJ, 130, 2363) and Reach et al. (2007, Icarus, 191, 298). Conclusions: The albedo of 4015 Wilson-Harrington is low, consistent with those of comet nuclei and primitive C-, P-, D-type asteorids. We establish a rough lower limit for the thermal inertia of W-H of 60 Jm-2s-0.5 K-1 when it is at r = 1 AU, which is slightly over the limit of 30 Jm-2 s-0.5 K-1 derived by Groussin et al. (2009, Icarus, 199, 568) for the thermal inertia of the nucleus of comet 22P/Kopff.

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Fasciola hepatica:the effect of the microfilament inhibitor cytochalasin B on the ultrastructure of the adult fluke

The effect of the microfilament inhibitor cytochalasin B (10 and 100 micrograms/ml) on the ultrastructure of adult Fasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100 micrograms/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100 micrograms/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100 micrograms/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down "yolk" globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).

Fasciola hepatica:the effect of the microtubule inhibitors colchicine and tubulozole-C on the ultrastructure of the adult fluke

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of T1 secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

Structural basis for the nuclear import of the human androgen receptor

Ligand-dependent nuclear import is crucial for the function of the androgen receptor (AR) in both health and disease. The unliganded AR is retained in the cytoplasm but, on binding 5alpha-dihydrotestosterone, it translocates into the nucleus and alters transcription of its target genes. Nuclear import of AR is mediated by the nuclear import factor importin-alpha, which functions as a receptor that recognises and binds to specific nuclear localisation signal (NLS) motifs on cargo proteins. We show here that the AR binds to importin-alpha directly, albeit more weakly than the NLS of SV40 or nucleoplasmin. We describe the 2.6-angstroms-resolution crystal structure of the importin-alpha-AR-NLS complex, and show that the AR binds to the major NLS-binding site on importin-alpha in a manner different from most other NLSs. Finally, we have shown that pathological mutations within the NLS of AR that are associated with prostate cancer and androgen-insensitivity syndrome reduce the binding affinity to importin-alpha and, subsequently, retard nuclear import; surprisingly, however, the transcriptional activity of these mutants varies widely. Thus, in addition to its function in the nuclear import of AR, the NLS in the hinge region of AR has a separate, quite distinct role on transactivation, which becomes apparent once nuclear import has been achieved.

Appropriate complexity for the prediction of coastal and estuarine geomorphic behaviour at decadal to centennial scales

Coastal and estuarine landforms provide a physical template that not only accommodates diverse ecosystem functions and human activities, but also mediates flood and erosion risks that are expected to increase with climate change. In this paper, we explore some of the issues associated with the conceptualisation and modelling of coastal morphological change at time and space scales relevant to managers and policy makers. Firstly, we revisit the question of how to define the most appropriate scales at which to seek quantitative predictions of landform change within an age defined by human interference with natural sediment systems and by the prospect of significant changes in climate and ocean forcing. Secondly, we consider the theoretical bases and conceptual frameworks for determining which processes are most important at a given scale of interest and the related problem of how to translate this understanding into models that are computationally feasible, retain a sound physical basis and demonstrate useful predictive skill. In particular, we explore the limitations of a primary scale approach and the extent to which these can be resolved with reference to the concept of the coastal tract and application of systems theory. Thirdly, we consider the importance of different styles of landform change and the need to resolve not only incremental evolution of morphology but also changes in the qualitative dynamics of a system and/or its gross morphological configuration. The extreme complexity and spatially distributed nature of landform systems means that quantitative prediction of future changes must necessarily be approached through mechanistic modelling of some form or another. Geomorphology has increasingly embraced so-called ‘reduced complexity’ models as a means of moving from an essentially reductionist focus on the mechanics of sediment transport towards a more synthesist view of landform evolution. However, there is little consensus on exactly what constitutes a reduced complexity model and the term itself is both misleading and, arguably, unhelpful. Accordingly, we synthesise a set of requirements for what might be termed ‘appropriate complexity modelling’ of quantitative coastal morphological change at scales commensurate with contemporary management and policy-making requirements: 1) The system being studied must be bounded with reference to the time and space scales at which behaviours of interest emerge and/or scientific or management problems arise; 2) model complexity and comprehensiveness must be appropriate to the problem at hand; 3) modellers should seek a priori insights into what kind of behaviours are likely to be evident at the scale of interest and the extent to which the behavioural validity of a model may be constrained by its underlying assumptions and its comprehensiveness; 4) informed by qualitative insights into likely dynamic behaviour, models should then be formulated with a view to resolving critical state changes; and 5) meso-scale modelling of coastal morphological change should reflect critically on the role of modelling and its relation to the observable world.