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)

Anisotropy of uni-axial and bi-axial deformation behavior of pure Titanium after hydrostatic extrusion

Coarse-grained commercially pure (CP) Titanium is subjected to hydrostatic extrusion resulting in the formation of ultrafine lamellar-type microstructure having very strong fiber texture. Uni-axial tensile tests of longitudinal and transverse specimens are carried out to study anisotropy of uni-axial deformation behavior of hydrostatically extruded CP Titanium. Small punch testing of longitudinal and transverse specimens is performed to study the anisotropy of its bi-axial deformation behavior. It is demonstrated that there is significant anisotropy of both uni-axial and bi-axial deformation of CP Titanium after hydrostatic extrusion which is related to the specific microstructure and texture developed in the material during hydrostatic extrusion.This work was carried out in frames of the European project LIMEDU (FP7 ERA-NET MATERA+2009, Project No MATERA/ESM- 1889) funded by Fundacion MADRI+D and by the National Centre for Research and Development (Project NCBiR/ERA-NET MATERA+/03/ 2011)

In-situ formation of Ag nanoparticles in the MAO coating during the processing of cp-Ti

Silver nanoparticle (Ag-NP) containing antibacterial micro-arc oxidation (MAO) coatings have already been synthesized over titanium-based materials via the MAO process employed in silver acetate (AgC2H3O2) containing electrolyte. However, the way of incorporation and in-situ formation of Ag-NPs within the MAO coating have not been documented yet. Present work was initiated to reveal the mechanism of Ag-NP formation within the MAO coatings. Thus, the structure of the MAO coating fabricated on commercial purity titanium in the AgC2H3O2-containing electrolyte was investigated by electron microscopy techniques. To this end, the cross-sectional high-resolution electron microscopy studies were carried out on lamella cut out with the focused ion beam technique, and these investigations were backed by X-ray photoelectron spectroscopy measurements of chemical composition on the surface of the MAO coating. These studies revealed that Ag is dispersed in the form of nanoparticles throughout the coating and that a higher density was confirmed closer to the micro-pores

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

Digital subtraction radiographic analysis of the combination of bioabsorbable membrane and bovine morphogenetic protein pool in human periodontal infrabony defects

Objectives: This study assessed the bone density gain and its relationship with the periodontal clinical parameters in a case series of a regenerative therapy procedure. Material and Methods: Using a split-mouth study design, 10 pairs of infrabony defects from 15 patients were treated with a pool of bovine bone morphogenetic proteins associated with collagen membrane (test sites) or collagen membrane only (control sites). The periodontal healing was clinically and radiographically monitored for six months. Standardized presurgical and 6-month postoperative radiographs were digitized for digital subtraction analysis, which showed relative bone density gain in both groups of 0.034 ± 0.423 and 0.105 ± 0.423 in the test and control group, respectively (p>0.05). Results: As regards the area size of bone density change, the influence of the therapy was detected in 2.5 mm2 in the test group and 2 mm2 in the control group (p>0.05). Additionally, no correlation was observed between the favorable clinical results and the bone density gain measured by digital subtraction radiography (p>0.05). Conclusions: The findings of this study suggest that the clinical benefit of the regenerative therapy observed did not come with significant bone density gains. Long-term evaluation may lead to a different conclusions

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

Application of the ultra-high materials obtained by hydrostatic extrusion in modern industry

W pracy przedstawiono ogólną charakterystykę wysokociśnieniowej przeróbki plastycznej metali i stopów metali metodą wyciskania hydrostatycznego. Opisano zalety procesu ze szczególnym uwzględnieniem możliwości rozdrabniania struktury metali i stopów metali do poziomu nanometrycznego lub ultradrobnoziarnistego, skutkującego znacznym podniesieniem ich właściwości mechanicznych. Pokazano przykłady praktycznego zastosowania przerabianych plastycznie metali, tj.: stali austenitycznej 316L na elementy złączne, miedzi stopowej CuCrZr na elektrody do procesu zgrzewania punktowego oraz tytanu do zastosowania na implanty medyczne.The general characteristic of the metals and alloys plastic deformation under high pressure by hydrostatic extrusion processing is presented. The advantages of the process, with special focus on the structural grain refinement to the ultrafine and nanocrystalline grain size levels resulting in significant increase of the material strength are described. Examples of the commercial application of working materials, as the 316L austenitic stainless steel for fixing elements, the CuCrZr copper alloy for spot welding electrodes and commercial purity titanium for medical implants are demonstrated