Curvature-coupling dependence of membrane protein diffusion coefficients

We consider the lateral diffusion of a protein interacting with the curvature of the membrane. The interaction energy is minimized if the particle is at a membrane position with a certain curvature that agrees with the spontaneous curvature of the particle. We employ stochastic simulations that take into account both the thermal fluctuations of the membrane and the diffusive behavior of the particle. In this study we neglect the influence of the particle on the membrane dynamics, thus the membrane dynamics agrees with that of a freely fluctuating membrane. Overall, we find that this curvature-coupling substantially enhances the diffusion coefficient. We compare the ratio of the projected or measured diffusion coefficient and the free intramembrane diffusion coefficient, which is a parameter of the simulations, with analytical results that rely on several approximations. We find that the simulations always lead to a somewhat smaller diffusion coefficient than our analytical approach. A detailed study of the correlations of the forces acting on the particle indicates that the diffusing inclusion tries to follow favorable positions on the membrane, such that forces along the trajectory are on average smaller than they would be for random particle positions.Comment: 16 pages, 8 figure

Helical Chirality: a Link between Local Interactions and Global Topology in DNA

DNA supercoiling plays a major role in many cellular functions. The global DNA conformation is however intimately linked to local DNA-DNA interactions influencing both the physical properties and the biological functions of the supercoiled molecule. Juxtaposition of DNA double helices in ubiquitous crossover arrangements participates in multiple functions such as recombination, gene regulation and DNA packaging. However, little is currently known about how the structure and stability of direct DNA-DNA interactions influence the topological state of DNA. Here, a crystallographic analysis shows that due to the intrinsic helical chirality of DNA, crossovers of opposite handedness exhibit markedly different geometries. While right-handed crossovers are self-fitted by sequence-specific groove-backbone interaction and bridging Mg2+ sites, left-handed crossovers are juxtaposed by groove-groove interaction. Our previous calculations have shown that the different geometries result in differential stabilisation in solution, in the presence of divalent cations. The present study reveals that the various topological states of the cell are associated with different inter-segmental interactions. While the unstable left-handed crossovers are exclusively formed in negatively supercoiled DNA, stable right-handed crossovers constitute the local signature of an unusual topological state in the cell, such as the positively supercoiled or relaxed DNA. These findings not only provide a simple mechanism for locally sensing the DNA topology but also lead to the prediction that, due to their different tertiary intra-molecular interactions, supercoiled molecules of opposite signs must display markedly different physical properties. Sticky inter-segmental interactions in positively supercoiled or relaxed DNA are expected to greatly slow down the slithering dynamics of DNA. We therefore suggest that the intrinsic helical chirality of DNA may have oriented the early evolutionary choices for DNA topology

Differences in lateral gene transfer in hypersaline versus thermal environments

<p>Abstract</p> <p>Background</p> <p>The role of lateral gene transfer (LGT) in the evolution of microorganisms is only beginning to be understood. While most LGT events occur between closely related individuals, inter-phylum and inter-domain LGT events are not uncommon. These distant transfer events offer potentially greater fitness advantages and it is for this reason that these "long distance" LGT events may have significantly impacted the evolution of microbes. One mechanism driving distant LGT events is microbial transformation. Theoretically, transformative events can occur between any two species provided that the DNA of one enters the habitat of the other. Two categories of microorganisms that are well-known for LGT are the thermophiles and halophiles.</p> <p>Results</p> <p>We identified potential inter-class LGT events into both a thermophilic class of Archaea (Thermoprotei) and a halophilic class of Archaea (Halobacteria). We then categorized these LGT genes as originating in thermophiles and halophiles respectively. While more than 68% of transfer events into Thermoprotei taxa originated in other thermophiles, less than 11% of transfer events into Halobacteria taxa originated in other halophiles.</p> <p>Conclusions</p> <p>Our results suggest that there is a fundamental difference between LGT in thermophiles and halophiles. We theorize that the difference lies in the different natures of the environments. While DNA degrades rapidly in thermal environments due to temperature-driven denaturization, hypersaline environments are adept at preserving DNA. Furthermore, most hypersaline environments, as topographical minima, are natural collectors of cellular debris. Thus halophiles would in theory be exposed to a greater diversity and quantity of extracellular DNA than thermophiles.</p

The evolution of bicontinuous polymeric nanospheres in aqueous solution

Complex polymeric nanospheres in aqueous solution are desirable for their promising potential in encapsulation and templating applications. Understanding how they evolve in solution enables better control of the final structures. By unifying insights from cryoTEM and small angle X-ray scattering (SAXS), we present a mechanism for the development of bicontinuous polymeric nanospheres (BPNs) in aqueous solution from a semi-crystalline comb-like block copolymer that possesses temperature-responsive functionality. During the initial stages of water addition to THF solutions of the copolymer the aggregates are predominantly vesicles; but above a water content of 53% irregular aggregates of phase separated material appear, often microns in diameter and of indeterminate shape. We also observe a cononsolvency regime for the copolymer in THF–water mixtures from 22 to 36%. The structured large aggregates gradually decrease in size throughout dialysis, and the BPNs only appear upon cooling the fully aqueous dispersions from 35 °C to 5 °C. Thus, the final BPNs are ultimately the result of a reversible temperature-induced morphological transition

Accelerating Drug Development Using Biomarkers: A Case Study with Sitagliptin, A Novel DPP4 Inhibitor for Type 2 Diabetes

The leveraged use of biomarkers presents an opportunity in understanding target engagement and disease impact while accelerating drug development. For effective integration in drug development, it is essential for biomarkers to aid in the elucidation of mechanisms of action and disease progression. The recent years have witnessed significant progress in biomarker selection, validation, and qualification, while enabling surrogate and clinical endpoint qualification and application. Biomarkers play a central role in target validation for novel mechanisms. They also play a central role in the learning/confirming paradigm, particularly when utilized in concert with pharmacokinetic/pharmacodynamic modeling. Clearly, these attributes make biomarker integration attractive for scientific and regulatory applications to new drug development. In this review, applications of proximal, or target engagement, and distal, or disease-related, biomarkers are highlighted using the example of the recent development of sitagliptin for type 2 diabetes, wherein elucidation of target engagement and disease-related biomarkers significantly accelerated sitagliptin drug development. Importantly, use of biomarkers as tools facilitated design of clinical efficacy trials while streamlining dose focus and optimization, the net impact of which reduced overall cycle time to filing as compared to the industry average

Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

International audienceWe introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells

Respiratory syncytial virus infection induces higher Toll-like receptor-3 expression and TNF-α production than human metapneumovirus infection

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Staphylococcus aureus enterotoxins induce IL-8 secretion by human nasal epithelial cells

BACKGROUND: Staphylococcus aureus produces a set of proteins which act both as superantigens and toxins. Although their mode of action as superantigens is well understood, little is known about their effects on airway epithelial cells. METHODS: To investigate this problem, primary nasal epithelial cells derived from normal and asthmatic subjects were stimulated with staphylococcal enterotoxin A and B (SEA and SEB) and secreted (supernatants) and cell-associated (cell lysates) IL-8, TNF-α, RANTES and eotaxin were determined by specific ELISAs. RESULTS: Non-toxic concentrations of SEA and SEB (0.01 μg/ml and 1.0 μg/ml) induced IL-8 secretion after 24 h of culture. Pre-treatment of the cells with IFN-γ (50 IU/ml) resulted in a further increase of IL-8 secretion. In cells from healthy donors pretreated with IFN-γ, SEA at 1.0 μg/ml induced release of 1009 pg/ml IL-8 (733.0–1216 pg/ml, median (range)) while in cells from asthmatic donors the same treatment induced significantly higher IL-8 secretion – 1550 pg/ml (1168.0–2000.0 pg/ml p = 0.04). Normal cells pre-treated with IFN-γ and then cultured with SEB at 1.0 μg/ml released 904.6 pg/ml IL-8 (666.5–1169.0 pg/ml). Cells from asthmatics treated in the same way produced significantly higher amounts of IL-8 – 1665.0 pg/ml (1168.0–2000.0 pg/ml, p = 0.01). Blocking antibodies to MHC class II molecules added to cultures stimulated with SEA and SEB, reduced IL-8 secretion by about 40% in IFN-γ unstimulated cultures and 75% in IFN-γ stimulated cultures. No secretion of TNF-α, RANTES and eotaxin was noted. CONCLUSION: Staphylococcal enterotoxins may have a role in the pathogenesis of asthma

Mechanisms Underlying the Confined Diffusion of Cholera Toxin B-Subunit in Intact Cell Membranes

Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM1 in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM1 complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes