Elastic continuum stiffness of contractile tail sheaths from molecular dynamics simulations.

Contractile tails are key components of the biological nanomachinery involved in cell membrane puncturing, where they provide a means to deliver molecules and ions inside cells. Two intriguing examples of contractile tails are those from bacteriophage T4 and R2-pyocin. Although the two systems are different in terms of biological activity, they share a fascinatingly similar injection mechanism, during which the tail sheaths of both systems contract from a so-called extended state to around half of their length (the contracted state), accompanied by release of elastic energy originally stored in the sheath. Despite the great prevalence and biomedical importance of contractile delivery systems, many fundamental details of their injection machinery and dynamics are still unknown. In this work, we calculate the bending and torsional stiffness constants of a helical tail sheath strand of bacteriophage T4 and R2-pyocin, in both extended and contracted states, using molecular dynamics simulations of about one-sixth of the entire sheath. Differences in stiffness constants between the two systems are rationalized by comparing their all-atom monomer structures, changes in sheath architecture on contraction, and differences in interstrand interactions. The calculated coefficients indicate that the T4 strand is stiffer for both bending and torsion than the corresponding R2-pyocin strands in both extended and contracted conformations. The sheath strands also have greater stiffness in the contracted state for both systems. As the main application of this study, we describe how the stiffness constants can be incorporated in a model to simulate the dynamics of contractile nanoinjection machineries.

DNA buckling in bacteriophage cavities as a mechanism to aid virus assembly.

While relatively simple biologically, bacteriophages are sophisticated biochemical machines that execute a precise sequence of events during virus assembly, DNA packaging, and ejection. These stages of the viral life cycle require intricate coordination of viral components whose structures are being revealed by single molecule experiments and high resolution (cryo-electron microscopy) reconstructions. For example, during packaging, bacteriophages employ some of the strongest known molecular motors to package DNA against increasing pressure within the viral capsid shell. Located upstream of the motor is an elaborate portal system through which DNA is threaded. A high resolution reconstruction of the portal system for bacteriophage ϕ29 reveals that DNA buckles inside a small cavity under large compressive forces. In this study, we demonstrate that DNA can also buckle in other bacteriophages including T7 and P22. Using a computational rod model for DNA, we demonstrate that a DNA buckle can initiate and grow within the small confines of a cavity under biologically-attainable force levels. The forces of DNA-cavity contact and DNA-DNA electrostatic repulsion ultimately limit cavity filling. Despite conforming to very different cavity geometries, the buckled DNA within T7 and P22 exhibits near equal volumetric energy density (∼1kT/nm(3)) and energetic cost of packaging (∼22kT). We hypothesize that a DNA buckle creates large forces on the cavity interior to signal the conformational changes to end packaging. In addition, a DNA buckle may help retain the genome prior to tail assembly through significantly increased contact area with the portal.

Structural ensemble and dynamics of toroidal-like DNA shapes in bacteriophage ϕ29 exit cavity.

In the bacteriophage ϕ29, DNA is packed into a preassembled capsid from which it ejects under high pressure. A recent cryo-EM reconstruction of ϕ29 revealed a compact toroidal DNA structure (30-40 basepairs) lodged within the exit cavity formed by the connector-lower collar protein complex. Using multiscale models, we compute a detailed structural ensemble of intriguing DNA toroids of various lengths, all highly compatible with experimental observations. In particular, coarse-grained (elastic rod) and atomistic (molecular dynamics) models predict the formation of DNA toroids under significant compression, a largely unexplored state of DNA. Model predictions confirm that a biologically attainable compressive force of 25 pN sustains the toroid and yields DNA electron density maps highly consistent with the experimental reconstruction. The subsequent simulation of dynamic toroid ejection reveals large reactions on the connector that may signal genome release.

DNA modeling reveals an extended lac repressor conformation in classic in vitro binding assays.

Protein-mediated DNA looping, such as that induced by the lactose repressor (LacI) of Escherichia coli, is a well-known gene regulation mechanism. Although researchers have given considerable attention to DNA looping by LacI, many unanswered questions about this mechanism, including the role of protein flexibility, remain. Recent single-molecule observations suggest that the two DNA-binding domains of LacI are capable of splaying open about the tetramerization domain into an extended conformation. We hypothesized that if recent experiments were able to reveal the extended conformation, it is possible that such structures occurred in previous studies as well. In this study, we tested our hypothesis by reevaluating two classic in vitro binding assays using a computational rod model of DNA. The experiments and computations evaluate the looping of both linear DNA and supercoiled DNA minicircles over a broad range of DNA interoperator lengths. The computed energetic minima align well with the experimentally observed interoperator length for optimal loop stability. Of equal importance, the model reveals that the most stable loops for linear DNA occur when LacI adopts the extended conformation. In contrast, for DNA minicircles, optimal stability may arise from either the closed or the extended protein conformation depending on the degree of supercoiling and the interoperator length.

A multiscale dynamic model of DNA supercoil relaxation by topoisomerase IB.

In this study, we report what we believe to be the first multiscale simulation of the dynamic relaxation of DNA supercoils by human topoisomerase IB (topo IB). We leverage our previous molecular dynamics calculations of the free energy landscape describing the interaction between a short DNA fragment and topo IB. Herein, this landscape is used to prescribe boundary conditions for a computational, elastodynamic continuum rod model of a long length of supercoiled DNA. The rod model, which accounts for the nonlinear bending, twisting, and electrostatic interaction of the (negatively charged) DNA backbone, is extended to include the hydrodynamic drag induced by the surrounding physiological buffer. Simulations for a 200-bp-long DNA supercoil in complex with topo IB reveal a relaxation timescale of ∼0.1-1.0 µs. The relaxation follows a sequence of cascading reductions in the supercoil linking number (Lk), twist (Tw), and writhe (Wr) that follow companion cascading reductions in the supercoil elastic and electrostatic energies. The novel (to our knowledge) multiscale modeling method may enable simulations of the entire experimental setup that measures DNA supercoiling and relaxation via single molecule magnetic trapping.

Computational analysis of looping of a large family of highly bent DNA by LacI.

Sequence-dependent intrinsic curvature of DNA influences looping by regulatory proteins such as LacI and NtrC. Curvature can enhance stability and control shape, as observed in LacI loops formed with three designed sequences with operators bracketing an A-tract bend. We explore geometric, topological, and energetic effects of curvature with an analysis of a family of highly bent sequences, using the elastic rod model from previous work. A unifying straight-helical-straight representation uses two phasing parameters to describe sequences composed of two straight segments that flank a common helically supercoiled segment. We exercise the rod model over this two-dimensional space of phasing parameters to evaluate looping behaviors. This design space is found to comprise two subspaces that prefer parallel versus anti-parallel binding topologies. The energetic cost of looping varies from 4 to 12 kT. Molecules can be designed to yield distinct binding topologies as well as hyperstable or hypostable loops and potentially loops that can switch conformations. Loop switching could be a mechanism for control of gene expression. Model predictions for linking numbers and sizes of LacI-DNA loops can be tested using multiple experimental approaches, which coupled with theory could address whether proteins or DNA provide the observed flexibility of protein-DNA loops.

Intrinsic curvature of DNA influences LacR-mediated looping.

Protein-mediated DNA looping is a common mechanism for regulating gene expression. Loops occur when a protein binds to two operators on the same DNA molecule. The probability of looping is controlled, in part, by the basepair sequence of inter-operator DNA, which influences its structural properties. One structural property is the intrinsic or stress-free curvature. In this article, we explore the influence of sequence-dependent intrinsic curvature by exercising a computational rod model for the inter-operator DNA as applied to looping of the LacR-DNA complex. Starting with known sequences for the inter-operator DNA, we first compute the intrinsic curvature of the helical axis as input to the rod model. The crystal structure of the LacR (with bound operators) then defines the requisite boundary conditions needed for the dynamic rod model that predicts the energetics and topology of the intervening DNA loop. A major contribution of this model is its ability to predict a broad range of published experimental data for highly bent (designed) sequences. The model successfully predicts the loop topologies known from fluorescence resonance energy transfer measurements, the linking number distribution known from cyclization assays with the LacR-DNA complex, the relative loop stability known from competition assays, and the relative loop size known from gel mobility assays. In addition, the computations reveal that highly curved sequences tend to lower the energetic cost of loop formation, widen the energy distribution among stable and meta-stable looped states, and substantially alter loop topology. The inclusion of sequence-dependent intrinsic curvature also leads to nonuniform twist and necessitates consideration of eight distinct binding topologies from the known crystal structure of the LacR-DNA complex.

An investigation of the solubility of various compounds in the hydrofluoroalkane propellants and possible model liquid propellants.

The aims of this study were to investigate descriptive parameters that may predict the solubility of compounds in the hydrofluoroalkane (HFA) propellants and to identify a model HFA propellant that is liquid at room temperature and atmospheric pressure. The solubility of 32 and 20 compounds chosen to give a wide range of physicochemical properties in HFA-134a and HFA-227, respectively, was measured. The Fedors solubility parameter and a computed log octanol water partition coefficient (CLOGP) were compared with the compounds' solubility in the HFA propellants. A total of 19 and 15 solutes had finite solubilities for HFA-134a and HFA-227, respectively, although the remaining solutes were miscible in all proportions. There was no apparent relation between solubility in HFA and the Fedors solubility parameter. This was not improved by considering the hydrogen-bonding potential of the compounds. When log solubility versus CLOGP was plotted, there was a linear relation for 16 and 12 of the compounds exhibiting a finite solubility in the HFA propellants, although four solutes (phenols) were displaced to the left of the linear relation. The remaining 3 compounds had much lower solubilities than was predicted from their CLOGPs, possibly as a consequence of their crystallinity (high melting points). Of the putative model propellants investigated (i.e., perfluorohexane (PFH), 1H-perfluorohexane [1H-PFH], and 2,2,2-trifluoroethanol), 1H-PFH was the most promising, with a linear relation between solubility in 1H-PFH and solubility in HFA propellant being observed. The solubilities in 1H-PFH were approximately 11 and 26% of those in HFA-134a and HFA-227.

In vitro dermal and transdermal delivery of doxycycline from ethanol/migliol 840 vehicles.

This work investigated the feasibility of dermal and transdermal delivery of doxycycline from vehicles containing Migliol 840 (M840) and ethanol. Delivery of the drug via the skin would provide a useful alternative to oral delivery, which has many undesirable side-effects, such as oesophageal ulceration and disturbance of the normal gut flora. Potential applications include malaria prophylaxis, and the treatment of acne vulgaris, Lyme disease and Reiter syndrome. Experiments were performed to determine the permeation of doxycycline across excised full-thickness human skin and heat-separated epidermal membranes from saturated solutions in ethanol, 1:1 and 2:1 ethanol/M840. Unusual burst behaviour was observed using an ethanol vehicle, possibly as a result of the formation of dimers at saturation. Doxycycline permeated to a higher degree from ethanolic vehicles when M840 is present, suggesting that M840 is capable of enhancing the permeation of doxycycline. The flux across full-thickness skin was highest from a 2:1 ethanol:M840 vehicle (2.41 microg cm(-2) h(-1)), sufficient to deliver 282 microg l(-1) using an area of application of 30 cm(2). The data also produced unexpected results in that permeability across heat separated skin was an order of magnitude greater than across full-thickness skin (28.75 microg cm(-2) h(-1) for the 2:1 ethanol:M840 vehicle). Depth profiling indicated that the drug distributed quite evenly throughout the epidermis. The mean amount of doxycycline recovered from the epidermis at the end of a permeation experiment was 458.4 microg ml(-1). This was far higher than the volume of extractable lipid present in the same unit area, approximately 52.3 microg ml(-1) and indicated that a large proportion of the drug must have been located within the proteinaceous domain. The data therefore suggest (1) significant amounts of doxycycline can be administered into and across the skin; (2) M840 is a potentially useful enhancing vehicle; and (3) the transcellular route was of significance.

Prospective controlled study of gastrointestinal stapled anastomoses.

A controlled prospective study was carried out in a university-affiliated community hospital to evaluate the use of gastrointestinal staples compared with conventional sutures for anastomotic construction. The study included 100 randomized cases (50 sutured and 50 stapled) requiring anastomoses. Consecutive patients were accepted into the study, and no patients were excluded. There was no significant difference between the two groups in operating room time or the duration of postoperative hospitalization, nasogastric intubation or intravenous intubation. The complication rate was similar and comparable to previously published results. On three occasions, it was necessary during operation to convert from the use of staples to sutures when immediate disruption was noted at a gastroduodenal anastomosis.