Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (~2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ~50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized λ DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2)

Thermo – mechanical properties of SPS produced self-healing thermal barrier coatings containing pure and alloyed MoSi2 particles

Yttria – partially stabilised zirconia (YPSZ) MoSi2 composites have been designed to prolong the lifetime of the matrix by self – healing cracks during thermal cycling. The healing reaction at high temperatures is based on the decomposition of MoSi2, leading to a volumetrically expanding reaction product, which seals the crack. In this work, coefficient of thermal expansion (CTE) and the fracture toughness of composites containing MoSi2 particles, produced by spark plasma sintering (SPS) have been compared to conventional YPSZ. The CTE mismatch between YPSZ and MoSi2 was found to be small, implying that thermally induced mismatch stresses will be small and the composites have a similar CTE to conventional YPSZ. Fracture toughness was found not to be affected by the particles and showed similar values to unreinforced YPSZ. Cracks introduced by indentation have been shown neither to prefer, or avoid, the particles suggesting that such a composite system is capable of autonomously activating the self – healing reaction

Diffusion studies in magnetron sputter deposited silicon nitride films

In this work, silicon nitride coatings were deposited by magnetron sputtering onto float glass substrates and post-deposition annealed at 650°C for 5min. The structures and compositions of the coatings were investigated by X-ray diffraction, X-ray reflectometry, scanning electron microscopy and energy dispersive X-ray spectroscopy. Samples were then over-coated with silver and subjected to a second annealing process to initiate the diffusion of silver through the adjacent coating layers. Additional silicon nitride coatings were then deposited on selected samples to produce Si3N4/Ag/Si3N4/glass stacks, which were annealed at temperatures in the range 100-600°C. Ag and Na diffusion coefficients were then calculated from compositional profiles obtained from time of flight secondary ion mass spectrometry analysis. The coatings deposited in this study were found to have stoichiometric Si3N4 compositions and were amorphous after annealing. The diffusion rate of silver through these coatings was found to depend on annealing temperature and coating density and roughness, which in turn can be related to the deposition conditions. © 2013 Elsevier B.V

Functional Regeneration of Supraspinal Connections in a Patient With Transected Spinal Cord Following Transplantation of Bulbar Olfactory Ensheathing Cells With Peripheral Nerve Bridging

Treatment of patients sustaining a complete spinal cord injury remains an unsolved clinical problem because of the lack of spontaneous regeneration of injured central axons. A 38-year-old man sustained traumatic transection of the thoracic spinal cord at upper vertebral level Th9. At 21 months after injury, the patient presented symptoms of a clinically complete spinal cord injury (American Spinal Injury Association class A-ASIA A). One of the patient's olfactory bulbs was removed and used to derive a culture containing olfactory ensheathing cells and olfactory nerve fibroblasts. Following resection of the glial scar, the cultured cells were transplanted into the spinal cord stumps above and below the injury and the 8-mm gap bridged by four strips of autologous sural nerve. The patient underwent an intense pre- and postoperative neurorehabilitation program. No adverse effects were seen at 19 months postoperatively, and unexpectedly, the removal of the olfactory bulb did not lead to persistent unilateral anosmia. The patient improved from ASIA A to ASIA C. There was improved trunk stability, partial recovery of the voluntary movements of the lower extremities, and an increase of the muscle mass in the left thigh, as well as partial recovery of superficial and deep sensation. There was also some indication of improved visceral sensation and improved vascular autoregulation in the left lower limb. The pattern of recovery suggests functional regeneration of both efferent and afferent long-distance fibers. Imaging confirmed that the grafts had bridged the left side of the spinal cord, where the majority of the nerve grafts were implanted, and neurophysiological examinations confirmed the restitution of the integrity of the corticospinal tracts and the voluntary character of recorded muscle contractions. To our knowledge, this is the first clinical indication of the beneficial effects of transplanted autologous bulbar cells

Investigation of silver diffusion in TiO2/Ag/TiO2 coatings

Low emissivity (low-E) coatings consisting of dielectric/silver/dielectric multi-layer stacks are applied to large-area architectural glazing to reduce heat losses from buildings. In this work TiO2/Ag/TiO2 stacks were deposited onto soda-lime glass by pulsed DC reactive magnetron sputtering. The coatings were annealed in the range 100-600 C to study silver diffusion through neighbouring layers. Depth-profiling analysis was performed on these samples using time-of-flight secondary ion mass spectrometry and selected samples were also analysed by X-ray photoelectron spectroscopy and Rutherford backscattering spectrometry. Fick's second diffusion law was used to find diffusion coefficient values and to investigate the temperature dependence of silver diffusion. To investigate film morphology and composition, scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were performed. The purpose of this study is the requirement for the understanding of the issue of silver diffusion during annealing treatments used in glass fabrication and the results obtained show that silver diffuses through the adjacent layers in a stack during heat treatment. However, in the temperature range investigated, the diffusion rates did not follow an Arrhenius dependence. At higher temperatures and longer annealing times sodium also diffuses from the glass into the coating, replacing the silver between the titania layers. © 2013 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. All rights reserved

A Conformable High Temperature Nitride Coating for Ti Alloys

There are many applications including aeroengine design where one would like to operate Ti or its alloys at higher temperatures, but the threat of oxidation or fire remains a longstanding challenge. Here, we have designed a bilayer nitride coating for Ti and its alloys produced by magnetron sputter deposition of a SiAlN coating (1.2 μm thick) with a Mo interlayer. We have taken advantage of interdiffusion and inter-reaction at the interface during cyclic oxidation at 800°C to form a layered nitride coating system comprising: a SiAlN top layer, a TiN0.26 and Ti5Si3 mixed phase interlayer, and a Ti-Mo solid solution. The novel TiN0.26 interlayer exhibits adaptive conformability via mechanical twinning, thereby accommodating the thermal mismatch strain between the coating and substrate. This, along with high adhesion, confers excellent thermal cycling life with no cracking, spallation and oxidation of the coating evident after hundreds of hours of cyclic oxidation (>40 cycles) in air at 800°C. This work provides a design pathway for a new family of coatings displaying excellent adhesion, adaptive conformability and superior environmental protection for Ti alloys at high temperature

Comparison of the oxidation behavior of a zirconium nitride coating in water vapor and air at high temperature

The oxidation behavior of zirconium nitride coating in high-temperature water vapor and air environments was studied. The parabolic rate constant of ZrN oxidizing in the water vapor environment at 600 ℃ was approximately 100 times faster than that in air, due to the larger pores and greater number of cracks that were formed across ZrO2 oxide layer formed during the water vapor oxidation process than during the air oxidation process. A bilayer-structure ZrO2 with tetragonal ZrO2 near the ZrN/ZrO2 interface and monoclinic ZrO2 approaching the outer ZrO2 surface were formed in both cases. The lateral cracks across the ZrO2 scale were caused by volume expansion from the tetragonal ZrO2 phase to the monoclinic ZrO2 phase transition

Assessment of the prevalence of radix entomolaris and distolingual canal in mandibular first molars in 15 countries: a multinational cross-sectional study with meta-analysis

Aim: The aim of this study was two-folded: i) to assess the prevalence of Distolingual Canal (DLC) and Radix Entomolaris (RE) in Mandibular First Molars (M1Ms), using Cone Beam Computed Tomography (CBCT) images and ii) to assess the impact of sociodemographic factors on the prevalence of these conditions worldwide. Methods: CBCT images were scanned retrospectively and the ones including bilateral M1Ms were included in the study. The evaluation was performed by 1 researcher in each country, trained with CBCT technology. A written and video instruction program explaining the protocol to be followed step-by-step was provided to all observers to calibrate them. The CBCT imaging screening procedure consisted of evaluating axial sections from coronal to apical. The presence of DLC and RE in M1Ms (yes/no) was identified and recorded. Results: Six thousand three hundred four CBCTs, representing 12,608 M1Ms, were evaluated. A significant difference was found between countries regarding the prevalence of both RE and DLC (P .05). Conclusion: The overall prevalence of RE and DLC in M1Ms was 3% and 22%. Additionally, both RE and DLC showed substantial bilaterally. These variations should be considered by endodontic clinicians during endodontic procedures in order to avoid potential complications

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity