Managing DNA polymerases: Coordinating DNA replication, DNA repair, and DNA recombination

Two important and timely questions with respect to DNA replication, DNA recombination, and DNA repair are: (i) what controls which DNA polymerase gains access to a particular primer-terminus, and (ii) what determines whether a DNA polymerase hands off its DNA substrate to either a different DNA polymerase or to a different protein(s) for the completion of the specific biological process? These questions have taken on added importance in light of the fact that the number of known template-dependent DNA polymerases in both eukaryotes and in prokaryotes has grown tremendously in the past two years. Most notably, the current list now includes a completely new family of enzymes that are capable of replicating imperfect DNA templates. This UmuC-DinB-Rad30-Rev1 superfamily of DNA polymerases has members in all three kingdoms of life. Members of this family have recently received a great deal of attention due to the roles they play in translesion DNA synthesis (TLS), the potentially mutagenic replication over DNA lesions that act as potent blocks to continued replication catalyzed by replicative DNA polymerases. Here, we have attempted to summarize our current understanding of the regulation of action of DNA polymerases with respect to their roles in DNA replication, TLS, DNA repair, DNA recombination, and cell cycle progression. In particular, we discuss these issues in the context of the Gram-negative bacterium, Escherichia coli, that contains a DNA polymerase (Pol V) known to participate in most, if not all, of these processes.

Roles of DNA polymerases V and II in SOS-induced error-prone and error-free repair in Escherichia coli

DNA polymerase V, composed of a heterotrimer of the DNA damage-inducible UmuC and UmuD\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{^{\prime}}}}\end{equation*}\end{document} proteins, working in conjunction with RecA, single-stranded DNA (ssDNA)-binding protein (SSB), β sliding clamp, and γ clamp loading complex, are responsible for most SOS lesion-targeted mutations in Escherichia coli, by catalyzing translesion synthesis (TLS). DNA polymerase II, the product of the damage-inducible polB (dinA ) gene plays a pivotal role in replication-restart, a process that bypasses DNA damage in an error-free manner. Replication-restart takes place almost immediately after the DNA is damaged (≈2 min post-UV irradiation), whereas TLS occurs after pol V is induced ≈50 min later. We discuss recent data for pol V-catalyzed TLS and pol II-catalyzed replication-restart. Specific roles during TLS for pol V and each of its accessory factors have been recently determined. Although the precise molecular mechanism of pol II-dependent replication-restart remains to be elucidated, it has recently been shown to operate in conjunction with RecFOR and PriA proteins.

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.

Células-tronco de membrana amniótica de cão como terapia alternativa para o tratamento de ceratoconjuntivite seca em cães

A Ceratoconjuntivite Seca (KCS Keratoconjunctivitis Sicca) é uma desordem imunomediada e resulta de alterações do componente aquoso do filme lacrimal e da deficiência dos componentes lipídicos e mucoso.Seu diagnóstico é baseado no Teste Lacrimal de Schirmer (TLS) e no Teste de Ruptura do Filme Lacrimal (TRFL) e tem como sinais clínicos: secreção mucopurulenta, hiperemia conjuntival, blefaroespasmos, fotofobia, incômodo, dor, vascularização, opacidade corneana e pigmentação, além de cegueira em casos avançados. O tratamento convencional consiste em aplicações diárias de Ciclosporina 0,2% ou Tacrolimus 0,03% (pomada ou colírio oftálmicos), que apesar de controlar a doença, são custosos, não curativos e exigem alto comprometimento da interação paciente-proprietário. A terapia celular usando células-tronco (CT) traz uma nova esperança para doenças sem tratamento efetivo. Neste trabalho utilizamos CT mesenquimais (CTM) obtidas a partir de membrana amniótica (CTMA) de cães obtidas a partir do descarte destes tecidos em campanhas de castrações em diferentes tempos gestacionais, sem formação tumoral quando submetidas ao teste tumorigênico durante 60 dias. Dois animais com KCS crônica foram tratados com duas injeções de CTMA com intervalo de 30 dias, sendo a primeira de 0,5x106 células e a segunda de 1x106 células em cada glândula. Na segunda semana após a terapia foi observado aumento da TLS sugerindo um benéficio da terapia que foi diminuindo com o passar das semanas. O TRFL oscilou durante os testes e não apresentou diferenças significativas. A terapia celular utilizando CTMA de cães melhorou a condição ocular nos dois casos em momentos e parâmetros variados, com repercussão na melhoria da superfície, mas não houve regressão do quadro clínico. Investigações futuras em estágios menos avançados da doença podem ajudar a elucidar os mecanismos pelos quais esse efeito foi obtido

Discontinuity Set Extractor

Disponible en Github: https://github.com/adririquelme/DSE

The story of a southern school; the Episcopal high school of Virginia,

Mode of access: Internet.

Digital surface model, hillshade and orthophoto of the world's largest fossil oyster reef, links to GeoTIFFs

The world's largest fossil oyster reef, formed by the giant oyster Crassostrea gryphoides and located in Stetten (north of Vienna, Austria) is studied by Harzhauser et al., 2015, 2016; Djuricic et al., 2016. Digital documentation of the unique geological site is provided by terrestrial laser scanning (TLS) at the millimeter scale. Obtaining meaningful results is not merely a matter of data acquisition with a suitable device; it requires proper planning, data management, and postprocessing. Terrestrial laser scanning technology has a high potential for providing precise 3D mapping that serves as the basis for automatic object detection in different scenarios; however, it faces challenges in the presence of large amounts of data and the irregular geometry of an oyster reef. We provide a detailed description of the techniques and strategy used for data collection and processing in Djuricic et al., 2016. The use of laser scanning provided the ability to measure surface points of 46,840 (estimated) shells. They are up to 60-cm-long oyster specimens, and their surfaces are modeled with a high accuracy of 1 mm. In addition to laser scanning measurements, more than 300 photographs were captured, and an orthophoto mosaic was generated with a ground sampling distance (GSD) of 0.5 mm. This high-resolution 3D information and the photographic texture serve as the basis for ongoing and future geological and paleontological analyses. Moreover, they provide unprecedented documentation for conservation issues at a unique natural heritage site.

Using Terrestrial LiDAR to Monitor Erosion within the Gold Basin Landslide Complex, Verlot, WA

In 2014 the United States Forest Service closed the Gold Basin Campground of western Washington in an effort to protect the public from unstable hillslopes directly adjacent to the campground. The Gold Basin Landslide Complex (GBLC) is actively eroding via block fall, dry ravel, and debris flows, which contribute sediment into the South Fork of the Stillaguamish River. This sediment diminishes the salmonid population within the South Fork of the Stillaguamish River by reducing habitable spawning grounds, which is a big concern to the Stillaguamish Tribe of Indians. In this investigation, I quantified patterns of degradation and total volume of sediment erosion from the middle lobe of the GBLC over the period of July 2015 through January 2016 using terrestrial (ground-based) LiDAR (TLS). I characterized site specific stratigraphy and geomorphic processes, and laid the groundwork for future, long-term monitoring of this site. Results of this investigation determined that ~ 4,800m3 of sediment was eroded from the middle lobe of the GBLC during the 6 month study period (July 2015 – January 2016). This erosion likely occurred from debris flows, raveling of poorly sorted sand and gravel deposits and block failures of high plasticity silts and clays, and/or other mass wasting mechanisms. The generalized stratigraphic sequence in the GBLC consists of alternating massive beds of sand and gravel with silts and clays. The low permeability of these silts and clays provide a perfect venue for groundwater to percolate, as I observed during field investigations, which likely contributes to the active instability of the hillslopes. Continued monitoring and mapping of this complex will lead to viable information that could help both the United States Forest Service and the Stillaguamish Tribe.

Calculating the Uncertainty of a Structure from Motion (SfM) Model, Cadman Quarry, Monroe, Washington

Structure from Motion (SfM) is a new form of photogrammetry that automates the rendering of georeferenced 3D models of objects using digital photographs and independently surveyed Ground Control Points (GCPs). This project seeks to quantify the error found in Digital Elevation Models (DEMs) produced using SfM. I modeled a rockslide found at the Cadman Quarry (Monroe, Washington) because the surface is vegetation-free, which is ideal for SfM and Terrestrial LiDAR Scanner (TLS) surveys. By using SfM, TLS, and GPS positioning at the same time, I attempted to find the deviation in the SfM model from the TLS model and GPS points. Using the deviation, I found the Root-Mean-Square Error (RMSE) between the SfM DEM and GPS positions. The RMSE of the SfM model when compared to surveyed GPS points is 17cm. I propagated the uncertainty of the GPS points with the RMSE of the SfM model to find the uncertainty of the SfM model compared to the NAD 1984 datum. The uncertainty of the SfM model compared to the NAD 1984 is 27cm. This study did not produce a model from the TLS that had sufficient resolution on horizontal surfaces to compare to surveyed GPS points.

A comparison of denoising methods for X-ray fluoroscopic images

Fluoroscopic images exhibit severe signal-dependent quantum noise, due to the reduced X-ray dose involved in image formation, that is generally modelled as Poisson-distributed. However, image gray-level transformations, commonly applied by fluoroscopic device to enhance contrast, modify the noise statistics and the relationship between image noise variance and expected pixel intensity. Image denoising is essential to improve quality of fluoroscopic images and their clinical information content. Simple average filters are commonly employed in real-time processing, but they tend to blur edges and details. An extensive comparison of advanced denoising algorithms specifically designed for both signal-dependent noise (AAS, BM3Dc, HHM, TLS) and independent additive noise (AV, BM3D, K-SVD) was presented. Simulated test images degraded by various levels of Poisson quantum noise and real clinical fluoroscopic images were considered. Typical gray-level transformations (e.g. white compression) were also applied in order to evaluate their effect on the denoising algorithms. Performances of the algorithms were evaluated in terms of peak-signal-to-noise ratio (PSNR), signal-to-noise ratio (SNR), mean square error (MSE), structural similarity index (SSIM) and computational time. On average, the filters designed for signal-dependent noise provided better image restorations than those assuming additive white Gaussian noise (AWGN). Collaborative denoising strategy was found to be the most effective in denoising of both simulated and real data, also in the presence of image gray-level transformations. White compression, by inherently reducing the greater noise variance of brighter pixels, appeared to support denoising algorithms in performing more effectively. © 2012 Elsevier Ltd. All rights reserved.

Transporte longitudinal de sedimento na zona costeira de Natal/RN

The longshore sediment transport (LST) is determinant for the occurrence of morphological changes in coastal environments. Understanding their movement mechanisms and transport is an essential source of information for the project design and coastal management plans. This study aims to characterize, initially, the active hydrodynamic circulation in the study area, comprised of four beach sectors from the south coast of Natal, assessing the average annual LST obtained through three proven equations (CERC, Kamphuis and Bayram et al.), defining the best formulation for the study area in question, and analyze the seasonal variability and the decadal transport evolution. The coastal area selected for this work constitutes one of the main tourist corridors in the city, but has suffered serious damage resulting from associated effects of hydrodynamic forcings and their disorderly occupation. As a tool was used the Coastal Modelling System of Brazil (SMC-Brazil), which presents integrated a series of numerical models and a database, properly calibrated and validated for use in developing projects along the Brazilian coastline. The LST rates were obtained for 15 beach profiles distributed throughout the study area. Their extensions take into account the depth of closure calculated by Harllermeier equation, and regarding the physical properties of the sediment, typical values of sandy beaches were adopted, except for the average diameter, which was calculated through an optimization algorithm based on equilibrium profile formulation proposed by Dean. Overall, the results showed an intensification of hydrodynamic forcings under extreme sea wave conditions, especially along the headlands exist in the region. Among the analyzed equations, Bayram et al. was the most suitable for this type of application, with a predominant transport in the south-north direction and the highest rates within the order of 700.000 m3 /year to 2.000.000 m3 /year. The seasonal analysis also indicated a longitudinal transport predominance in the south to north, with the highest rates associated with the fall and winter seasons. In these periods are observed erosive beach states, which indicate a direct relationship between the sediment dynamics and the occurrence of more energetic sea states. Regarding the decadal evolution of transportation, it was found a decrease in transport rate from the 50’s to the 70’s, followed by an increase until the 2000’s, coinciding with the beginning of urbanization process in some stretches of the studied coastline.

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo.

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18(-/-) mice. Moreover, primary Rad18(-/-) mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18(-/-) HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18(-/-) mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting.

Building a Rockfall Database Using Remote Sensing: Techniques for Hazard Management in Canadian Rail Corridors

The section of CN railway between Vancouver and Kamloops runs along the base of many hazardous slopes, including the White Canyon, which is located just outside the town of Lytton, BC. The slope has a history of frequent rockfall activity, which presents a hazard to the railway below. Rockfall inventories can be used to understand the frequency-magnitude relationship of events on hazardous slopes, however it can be difficult to consistently and accurately identify rockfall source zones and volumes on large slopes with frequent activity, leaving many inventories incomplete. We have studied this slope as a part of the Canadian Railway Ground Hazard Research Program and have collected remote sensing data, including terrestrial laser scanning (TLS), photographs, and photogrammetry data since 2012, and used change detection to identify rockfalls on the slope. The objective of this thesis is to use a subset of this data to understand how rockfalls identified from TLS data could be used to understand the frequency-magnitude relationship of rockfalls on the slope. This includes incorporating both new and existing methods to develop a semi-automated workflow to extract rockfall events from the TLS data. We show that these methods can be used to identify events as small as 0.01 m3 and that the duration between scans can have an effect on the frequency-magnitude relationship of the rockfalls. We also show that by incorporating photogrammetry data into our analysis, we can create a 3D geological model of the slope and use this to classify rockfalls by lithology, to further understand the rockfall failure patterns. When relating the rockfall activity to triggering factors, we found that the amount of precipitation occurring over the winter has an effect on the overall rockfall frequency for the remainder of the year. These results can provide the railways with a more complete inventory of events compared to records created through track inspection, or rockfall monitoring systems that are installed on the slope. In addition, we can use the database to understand the spatial and temporal distribution of events. The results can also be used as an input to rockfall modelling programs.

Confronto tra tecniche di remote sensing per la caratterizzazione di un ammasso roccioso presso le Gole di Scascoli, Loiano (BO)

Il versante sinistro delle Gole di Scascoli (BO) è caratterizzato da una marcata tendenza evolutiva per crollo e ribaltamento. Negli ultimi 25 anni si sono verificati eventi parossistici con volumi di roccia coinvolti rispettivamente di 7000 m3, 20000 m3 e 35000 m3. Il sito è di grande rilevanza a causa del forte fattore di rischio rappresentato per la strada di fondovalle ad esso adiacente. Il lavoro di tesi è stato finalizzato allo studio dei fenomeni di versante di una parete rocciosa inaccessibile nota in letteratura come “ex-Mammellone 1” mediante tecniche di telerilevamento quali TLS (Terrestrial Laser Scanning) e CRP (Close Range Photogrammetry) al fine affiancare il rilievo geomeccanico soggettivo dell’area svolto nel 2003 da ENSER Srl in seguito ai fenomeni di crollo del 2002. Lo sviluppo di tecnologie e metodi innovativi per l’analisi territoriale basata sull’impiego di UAV (Unmanned Aerial Vehicle, meglio noti come Droni), associata alle tecniche di fotogrammetria digitale costituisce un elemento di notevole ausilio nelle pratiche di rilevamento in campo di sicurezza e tempi di esecuzione. Il lavoro ha previsto una prima fase di rilevamento areo-fotogrammetrico mediante strumentazione professionale e amatoriale, a cui è seguita l’elaborazione dei rispettivi modelli. I diversi output sono stati confrontati dal punto di vista geomorfologico, geometrico, geomeccanico e di modellazione numerica di caduta massi. Dal lavoro è stato possibile indagare l’evoluzione morfologica del sito in esame negli ultimi 10 anni, confrontare diversi metodi di rilevamento e analisi dati, sperimentare la robustezza e ripetibilità geometrica del metodo fotogrammetrico per il rilievo di fronti rocciosi e mettere a punto un metodo semiautomatico di individuazione e analisi delle giaciture delle discontinuità.

Design and development of distributed feedback transistor lasers

The transistor laser is a unique three-port device that operates simultaneously as a transistor and a laser. With quantum wells incorporated in the base regions of heterojunction bipolar transistors, the transistor laser possesses advantageous characteristics of fast base spontaneous carrier lifetime, high differential optical gain, and electrical-optical characteristics for direct “read-out” of its optical properties. These devices have demonstrated many useful features such as high-speed optical transmission without the limitations of resonance, non-linear mixing, frequency multiplication, negative resistance, and photon-assisted switching. To date, all of these devices operate as multi-mode lasers without any type of wavelength selection or stabilizing mechanisms. Stable single-mode distributed feedback diode laser sources are important in many applications including spectroscopy, as pump sources for amplifiers and solid-state lasers, for use in coherent communication systems, and now as TLs potentially for integrated optoelectronics. The subject of this work is to expand the future applications of the transistor laser by demonstrating the theoretical background, process development and device design necessary to achieve singlelongitudinal- mode operation in a three-port transistor laser. A third-order distributed feedback surface grating is fabricated in the top emitter AlGaAs confining layers using soft photocurable nanoimprint lithography. The device produces continuous wave laser operation with a peak wavelength of 959.75 nm and threshold current of 13 mA operating at -70 °C. For devices with cleaved ends a side-mode suppression ratio greater than 25 dB has been achieved.