Novel ribonucleotide discrimination in the RNA polymerase-like two-barrel catalytic core of Family D DNA polymerases.

Family D DNA polymerase (PolD) is the essential replicative DNA polymerase for duplication of most archaeal genomes. PolD contains a unique two-barrel catalytic core absent from all other DNA polymerase families but found in RNA polymerases (RNAPs). While PolD has an ancestral RNA polymerase catalytic core, its active site has evolved the ability to discriminate against ribonucleotides. Until now, the mechanism evolved by PolD to prevent ribonucleotide incorporation was unknown. In all other DNA polymerase families, an active site steric gate residue prevents ribonucleotide incorporation. In this work, we identify two consensus active site acidic (a) and basic (b) motifs shared across the entire two-barrel nucleotide polymerase superfamily, and a nucleotide selectivity (s) motif specific to PolD versus RNAPs. A novel steric gate histidine residue (H931 in Thermococcus sp. 9°N PolD) in the PolD s-motif both prevents ribonucleotide incorporation and promotes efficient dNTP incorporation. Further, a PolD H931A steric gate mutant abolishes ribonucleotide discrimination and readily incorporates a variety of 2' modified nucleotides. Taken together, we construct the first putative nucleotide bound PolD active site model and provide structural and functional evidence for the emergence of DNA replication through the evolution of an ancestral RNAP two-barrel catalytic core.

RADAR-seq: A RAre DAmage and Repair sequencing method for detecting DNA damage on a genome-wide scale.

RAre DAmage and Repair sequencing (RADAR-seq) is a highly adaptable sequencing method that enables the identification and detection of rare DNA damage events for a wide variety of DNA lesions at single-molecule resolution on a genome-wide scale. In RADAR-seq, DNA lesions are replaced with a patch of modified bases that can be directly detected by Pacific Biosciences Single Molecule Real-Time (SMRT) sequencing. RADAR-seq enables dynamic detection over a wide range of DNA damage frequencies, including low physiological levels. Furthermore, without the need for DNA amplification and enrichment steps, RADAR-seq provides sequencing coverage of damaged and undamaged DNA across an entire genome. Here, we use RADAR-seq to measure the frequency and map the location of ribonucleotides in wild-type and RNaseH2-deficient E. coli and Thermococcus kodakarensis strains. Additionally, by tracking ribonucleotides incorporated during in vivo lagging strand DNA synthesis, we determined the replication initiation point in E. coli, and its relation to the origin of replication (oriC). RADAR-seq was also used to map cyclobutane pyrimidine dimers (CPDs) in Escherichia coli (E. coli) genomic DNA exposed to UV-radiation. On a broader scale, RADAR-seq can be applied to understand formation and repair of DNA damage, the correlation between DNA damage and disease initiation and progression, and complex biological pathways, including DNA replication.

Characterization and study of the antibacterial mechanisms of silver nanoparticles prepared with microalgal exopolysaccharides.

The green synthesis of biomaterials is of significant interest as it enables the safe and sustainable preparation of noble metallic nanoparticles for medical applications. Microalgae polysaccharides have received attention due to their outstanding properties such as biocompatibility, biodegradability and low cost. In addition, due to their variety of remarkable biological and physicochemical properties, polysaccharide-based nanoparticles have advantageous features yet to be explored. The primary objective of the current research was to investigate exopolysaccharides isolated from green microalgae Botryococcus braunii (EPBb) and Chlorella pyrenoidosa (EPCp), as both reducing and stabilizing agents, for the green synthesis of silver nanoparticles (AgNPs). Their antibacterial activity towards Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and antibiotic-resistant bacteria (methicillin-resistant Staphylococcus aureus) was studied, as well as their cytotoxicity to human dermal fibroblasts. The presently synthesized AgNPs were spherical in shape and exhibited characteristic surface plasmon resonance at 430 nm. The main population had a particle size which ranged between 5 and 15 nm as analyzed by transmission electron micrographs. Zeta potentials averaged -51.81 ±â€¯3.01 mV using EPBb and -12.16 ±â€¯2.41 mV using EPCp. More importantly, AgNPs possessed strong antibacterial activity in a dose-dependent manner, even against drug-resistant bacteria. The enhanced antibacterial activity of these particles is explained due to extensive reactive oxygen species generation and bacterial cell membrane damage. In contrast, such AgNPs were not cytotoxic at the same therapeutic range to fibroblasts (0.5-10.0 µg/mL). In summary, these results showed that polysaccharide-capped AgNPs have a strong potential for numerous medical applications, such as antibacterial agents in pharmaceutical and biomedical areas.

pH-Controlled Cerium Oxide Nanoparticle Inhibition of Both Gram-Positive and Gram-Negative Bacteria Growth.

Here, the antibacterial activity of dextran-coated nanoceria was examined against Pseudomonas aeruginosa and Staphylococcus epidermidis by varying the dose, the time of treatment, and the pH of the solution. Findings suggested that dextran-coated nanoceria particles were much more effective at killing P. aeruginosa and S. epidermidis at basic pH values (pH = 9) compared to acidic pH values (pH = 6) due to a smaller size and positive surface charge at pH 9. At pH 9, different particle concentrations did cause a delay in the growth of P. aeruginosa, whereas impressively S. epidermidis did not grow at all when treated with a 500 µg/mL nanoceria concentration for 24 hours. For both bacteria, a 2 log reduction and elevated amounts of reactive oxygen species (ROS) generation per colony were observed after 6 hours of treatment with nanoceria at pH 9 compared to untreated controls. After 6 hours of incubation with nanoceria at pH 9, P. aeruginosa showed drastic morphological changes as a result of cellular stress. In summary, this study provides significant evidence for the use of nanoceria (+4) for a wide range of anti-infection applications without resorting to the use of antibiotics, for which bacteria are developing a resistance towards anyway.

Conformation-mediated Förster resonance energy transfer (FRET) in blue-emitting polyvinylpyrrolidone (PVP)-passivated zinc oxide (ZnO) nanoparticles.

Homopolymers, such as polyvinylpyrrolidone (PVP), are commonly used to passivate the surface of blue-light emitting ZnO nanoparticles during colloid nucleation and growth. However, although PVP is known to auto-fluoresce at 400nm, which is near the absorption edge of ZnO, the impact of PVP adsorption characteristics on the surface of ZnO and the surface-related photophysics of PVP-capped ZnO nanoparticles is not well understood. To investigate, we have synthesized ZnO nanoparticles in solvents containing PVP of 3 concentrations-0.5, 0.7, and 0.11gmL-1. Using time-domain NMR, we show that the adsorbed polymer conformation differs with polymer concentration-head-to-tail under low concentration (e.g., 0.05gmL-1) and looping, then train-like, with increasing concentration (e.g., 0.07gmL-1 and 0.11gmL-1, respectively). When the surface-adsorbed PVP is entrained, the surface states of ZnO are passivated and radiative emission from surface trap states is suppressed, allowing emission to be dominated by exciton transitions in the UV (ca. 310nm). Moreover, the reduced proximity between the PVP molecule and the ZnO gives rise to increased efficiency of energy transfer between the exciton emission of ZnO and the HOMO-LUMO absorption of PVP (ca. 400nm). As a result, light emission in the blue is enhanced in the PVP-capped ZnO nanoparticles. We thus show that the emission properties of ZnO can be tuned by controlling the adsorbed PVP conformation on the ZnO surface via the PVP concentration in the ZnO precipitation medium.

pH-Dependent Activity of Dextran-Coated Cerium Oxide Nanoparticles on Prohibiting Osteosarcoma Cell Proliferation.

Cerium oxide nanoparticles (or nanoceria) have demonstrated great potential as antioxidants in various cell culture models. Despite such promise for reducing reactive oxygen species and an ability for surface functionalization, nanoceria has not been extensively studied for cancer applications to date. Herein, we engineered the surface of nanoceria with dextran and observed its activity in the presence bone cancer cells (osteosarcoma cells) at different pH values resembling the cancerous and noncancerous environment. We found that dextran coated nanoceria was much more effective at killing bone cancer cells at slightly acidic (pH 6) compared to physiological and basic pH values (pH 7 and pH 9). In contrast, minimal toxicity was observed for healthy (noncancerous) bone cells when cultured with nanoceria at pH = 6 after 1 day of treatment in the concentration range of 10-1000 µg/mL. Although healthy bone cancer cell viability decreased after treatment with high ceria nanoparticle concentrations (250-1000 µg/mL) for longer time periods at pH 6 (3 days and 5 days), approximately 2-3 fold higher healthy bone cell viabilities were observed compared to osteosarcoma cell viability at similar conditions. Very low toxicity was observed for healthy osteoblasts cultured with nanoceria for any concentration at any time period at pH 7. In this manner, this study provides the first evidence that nanoceria can be a promising nanoparticle for treating bone cancer without adversely affecting healthy bone cells and thus deserves further investigation.

Nanotechnology and picotechnology to increase tissue growth: a summary of in vivo studies.

The aim of tissue engineering is to develop functional substitutes for damaged tissues or malfunctioning organs. Since only nanomaterials can mimic the surface properties (ie, roughness) of natural tissues and have tunable properties (such as mechanical, magnetic, electrical, optical, and other properties), they are good candidates for increasing tissue growth, minimizing inflammation, and inhibiting infection. Recently, the use of nanomaterials in various tissue engineering applications has demonstrated improved tissue growth compared to what has been achieved until today with our conventional micron structured materials. This short report paper will summarize some of the more relevant advancements nanomaterials have made in regenerative medicine, specifically improving bone and bladder tissue growth. Moreover, this short report paper will also address the continued potential risks and toxicity concerns, which need to be accurately addressed by the use of nanomaterials. Lastly, this paper will emphasize a new field, picotechnology, in which researchers are altering electron distributions around atoms to promote surface energy to achieve similar increased tissue growth, decreased inflammation, and inhibited infection without potential nanomaterial toxicity concerns.

Diameter of titanium nanotubes influences anti-bacterial efficacy.

Bacterial infection of in-dwelling medical devices is a growing problem that cannot be treated by traditional antibiotics due to the increasing prevalence of antimicrobial resistance and biofilm formation. Here, due to changes in surface parameters, it is proposed that bacterial adhesion can be prevented through nanosurface modifications of the medical device alone. Toward this goal, titanium was created to possess nanotubular surface topographies of highly controlled diameters of 20, 40, 60, or 80 nm, sometimes followed by heat treatment to control chemistry and crystallinity, through a novel anodization process. For the first time it was found that through the control of Ti surface parameters including chemistry, crystallinity, nanotube size, and hydrophilicity, significantly changed responses of both Staphylococcus epidermidis and Staphylococcus aureus (pathogens relevant for orthopaedic and other medical device related infections) were measured. Specifically, heat treatment of 80 nm diameter titanium tubes produced the most robust antimicrobial effect of all surface treatment parameters tested. This study provides the first step toward understanding the surface properties of nano-structured titanium that improve tissue growth (as has been previously observed with nanotubular titanium), while simultaneously reducing infection without the use of pharmaceutical drugs.

Anodized 20 nm diameter nanotubular titanium for improved bladder stent applications.

Materials currently used for bladder applications often suffer from incomplete coverage by urothelial cells (cells that line the interior of the bladder and ureter) which leads to the continuous exposure of the underlying materials aggravating an immune response. In particular, a ureteral (or sometimes called an ureteric or bladder) stent is a thin tube inserted into the ureter to prevent or treat obstruction of urine flow from the kidney. The main complications with ureteral stents are infection and blockage by encrustation, which can be avoided by promoting the formation of a monolayer of urothelial cells on the surface of the stent. Nanotechnology (or the use of nanomaterials) may aid in urothelialization of bladder stents since nanomaterials have been shown to have unique surface energetics to promote the adsorption of proteins important for urothelial cell adhesion and proliferation. Since many bladder stents are composed of titanium, this study investigated the attachment and spreading of human urothelial cells on different nanotextured titanium surfaces. An inexpensive and effective scaled up anodization process was used to create equally distributed nanotubular surfaces of different diameter sizes from 20-80 nm on titanium with lengths approximately 500 nm. Results showed that compared to untreated titanium stents and 80 nm diameter nanotubular titanium, 20 nm diameter nanotubular titanium stents enhanced human urothelial cell adhesion and growth up to 3 days in culture. In this manner, this study suggests that titanium anodized to possess nanotubular surface features should be further explored for bladder stent applications.