Evaluating the genotoxic and cytotoxic effects of synthetic nucleosides in vitro

2019 Fall.Includes bibliographical references.The convoluted interplay between various cellular organelles has been a prominent area of study since humans have had the ability to research and explore the microscopic cellular world. Particularly, significant attention has been exercised in the effect that various compounds, pharmaceuticals, drugs, and therapies have on cellular division; particularly cancer cell division. Although documentation is scant, monitoring cell division has been of great interest for years. The utilization and administration of tritiated thymidine, to visualize cellular replication, was unarguably the first strategy to monitor cellular division. However, this method was deemed toxic and cumbersome. 5-bromo-2'-deoxyuridine (BrdU) soon took high notoriety. BrdU, a halogenated pyrimidine, and its structurally related analogs are known to mimic the deoxynucleoside, thymidine, during S-phase of cellular replication. BrdU is incorporated in place of thymidine during S-phase, and its rate of incorporation can be monitored via immunohistochemical antibody detection. However, current literature has demonstrated that BrdU presents a number of complications regarding long-term labeling, cell cycle progression, cellular mutagenicity and cytotoxicity, and unwanted photosensitivity. BrdU's shortcomings were bypassed by the advent of 5-ethynyl-2'-deoxyuridine (EdU) (25). EdU, an additional synthetic analog of thymidine, having a terminal 5'-ethynyl- substituent, instead of thymidine's or BrdU's terminal 5'-methyl- or 5'-bromo- substituent, respectively. EdU has gained popularity as the preferred method in detecting cellular division due to its inherent ability to readily incorporate into newly synthesized DNA. In order to detect and incorporate BrdU into DNA, the process requires the expenditure of an antibody. Binding of the BrdU-antibody tandem to DNA necessitates denaturation of DNA via volatile acid or heat treatment, which presents complications as unqualified and unfaithful base-paring and reannealing. Conversely, EdU incorporation and detection is a fast, simple, and effective method in labeling actively dividing cells. By way of "click" chemistry, EdU is readily introduced and synthesized into new DNA. The latter is accomplished, in part by a small-sized fluorescent azide, qualifying easy access to DNA without considerable steric hindrance. It is expected that successful incorporation of EdU, via "click" chemistry will result in high resolution microscopy analysis. However, current research suggests that implementation of EdU may result in unwanted biological effects. Using an in vitro system, the experimental basis described herein sought to determine the effects that BrdU or EdU had on cell cytotoxicity and genotoxicity when incorporated in DNA. Whilst a vast majority of research experiments use concentrations of said nucleosides' in the range of 10-50 µM, these conditions may induce strong genotoxic and cytotoxic effects inherently higher than the expected background frequency. By treating various DNA repair deficient cells with BrdU or EdU, at concentrations ranging from 1-100 µM, there was a significant increase in the induction of sister chromatid exchanges. Also, with identical concentrations as the latter, the doubling time of particular DNA repair deficient cell lines increased dramatically. To examine the effects of BrdU and EdU on DNA repair, a poly (ADP ribose) polymerase (PARP) ELISA assay was carried out. The PARP assay concluded that BrdU possessed the highest degree of PARP inhibition, with thymidine second, and EdU with the least PARP inhibition. One suggested mechanism by which BrdU is thought to implicate or hinder DNA repair is through its incorporation and modification of DNA repair thus, slower repair kinetics. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutation analysis suggests that manufacturers recommended EdU concentration (10µM), result in a significantly higher HPRT mutation frequency, compared to control. In addition to BrdU's SCE-induction capability and HPRT-induction incidence, clinical and radiotherapeutic properties have been examined. CHO cells exposed to 2 and 8 µM BrdU and 4 or 15 Gy X-rays, increase DNA repair duration, increased chromosomal fragmentation, and induce radiosensitization. However, little or no evidence is available in regard to EdU's propensity to affect cell viability. To assess the induction of cellular radiosensitivity and chromosomal aberrations, we investigated CHO and A549 (human lung cancer) cells replicative ability in the presence of three external radiation. An in vitro clonogenic and chromosomal aberration assay, in the presence of UVC-, photon (fluorescent)-, and γ-irradiation and BrdU or EdU, was implemented. Our results support BrdU's ability to decrease cell viability. Although each synthetic analogue presented their own biological contribution, their mechanism is still not fully understood. This study aims to discern any cytotoxic and/or genotoxic effects that EdU or BrdU pose on cell cycle progression, clonogenicity and viability, mutation-induction, chromosomal aberrations, and induction of radiosensitization

Archaeological Investigation of an Oil Well Pad Disturbance at the Tom Moore Site (41PN149), Panola County, Texas

The Tom Moore site (41PN149) is situated on the east slope of a circular-shaped landform at the highest point of a steeply-sloping upland in the Irons Bayou valley in Panola County, Texas. Irons Bayou, 1.2 km to the west of the site, flows east to its confluence with the Sabine River. A small tributary of Irons Bayou is 600 m to the south. Soils here are a brown sandy loam overlying a very hard red clay B-horizon. Like most of East Texas, the land has been farmed previously, as indicated by old plow furrows, and it has reforested naturally in pine and mixed hardwoods in the last 30 years. The site\u27s upland setting is similar to other Middle Caddoan sites in the Sabine River basin in East Texas. An oil field employee, J. W. Golden of Kilgore, Texas, located the Tom Moore site. He noticed a cache of seven large celts that had been disturbed by construction equipment preparing a well site. The oil well construction consisted of two wells and three associated storage tanks. Approximately 2 acres had been disturbed during the leveling process, and part of the hill was used to form the level well pad. A cultural resources management survey had not been required by the Railroad Commission of Texas prior to construction. Mr. Golden described the celts as occurring in a group with the blade ends up. The heavy equipment had grazed them and caused some damage. He collected the celts and informed the authors. We visited the site, made a surface collection, noted a midden area at one end of the disturbance, and collected soil samples for flotation (these materials were submitted to S. Eileen Goldborer of Paleoethnobotanical Services, Austin, Texas) as well as OCR dates. No testing was conducted by the authors to determine the extent of the site. A small, circular mound is located in the wooded area adjacent to the well pad

On Calculation of Thermal Conductivity from Einstein Relation in Equilibrium MD

In equilibrium molecular dynamics, Einstein relation can be used to calculate the thermal conductivity. This method is equivalent to Green-Kubo relation and it does not require a derivation of an analytical form for the heat current. However, it is not commonly used as Green-Kubo relationship. Its wide use is hindered by the lack of a proper definition for integrated heat current (energy moment) under periodic boundary conditions. In this paper, we developed an appropriate definition for integrated heat current to calculate thermal conductivity of solids under periodic conditions. We applied this method to solid argon and silicon based systems; compared and contrasted with the Green-Kubo approach.Comment: We updated this manuscript from second version by changing the title and abstract. This paper is submitted to J. Chem. Phy

\u3ci\u3eStachybotrys atra\u3c/i\u3e, an Effective Aggregator of Peorian Loess

Twelve cellulose-decomposing fungi were tested for aggregating ability with Peorian loess containing ground straw at a concentration of 1%. Of the 12 fungi, Stachybotrys atra was the most effective soil aggregator. Under laboratory conditions used, it produced from 2 to 30 times more aggregation than any of the other fungi tested. Studies were made of the influence of various environmental factors on the degree of aggregation effected by S. atra. An incubation time of 1 week was sufficient for a relatively high degree of aggregation. In comparison, longer periods resulted in only slightly improved aggregation. Varying the temperature between 20° and 28° C. had no appreciable effect on the aggregation by S. atra. Approximately equivalent aggregations were attained at the moisture levels of 20, 25, and 30%, which were definitely superior to the 10 and 15% levels. Alfalfa and straw, either separately or as a mixture, were satisfactory sources of energy material for S. atra. The aggregation obtained with alfalfa, however, was somewhat higher than that obtained with straw

Special Lagrangian cones with higher genus links

For every odd natural number g=2d+1 we prove the existence of a countably infinite family of special Lagrangian cones in C^3 over a closed Riemann surface of genus g, using a geometric PDE gluing method.Comment: 48 page

Kinetic analysis of microcalorimetric data derived from microbial growth: Basic theoretical, practical and industrial considerations

We report here a mathematical framework for the quantitative interpretation of exponential bacterial growth measured with isothermal microcalorimetry. The method allows determination of many parameters that define the exponential growth phase. To automate the analysis, we also wrote a coding program, so that the approach could be embedded in a commercial setting. As an exemplar, we apply the method to a commercial probiotic product. The outcome was that we could identify characteristic parameters of growth (including rate constant and doubling time), and hence authenticate product quality, within 15 h. This compares favourably with the current 7–10 days required for conventional microbiological assessment (to allow release of product for bottling and marketing) via plating methods. The method would lend itself to growth analysis of single and mixed bacterial cultures

Archaeological Investigations at the Redwine Site (41SM193), Smith County, Texas

The Redwine site (41SM193) is a probable Middle Caddoan habitation site located on an upland terrace (Figure I) on the headwaters of Auburn Creek, a small tributary of the Sabine River in central Smith County; the Angelina River drainage basin begins about 1.5 km to the south of the site. Auburn Creek is about 100 meters to the north of the site. The Sabine River lies approximately 24 km to the north. Soils on the Redwine site are Bowie fine sandy loam. The site was discovered in the early 1960s by Sam Whlteside an avocational archaeologist who lived in the Tyler area. His work consisted of trenching, and he located and excavated several burials and a small house mound. In an attempt to relocate the site limited controlled excavations were undertaken in 1995 by the authors, under the direction of Dr. John Keller of Southern Archaeological Consultants, Inc. We hoped to gain enough information about the size, age, and integrity of the Redwine site to apply for legal designation and protection under the Antiquities Code of Texas. After confirming that the Redwine site contained important archaeological information, an application for State Archeological Landmark (SAL) designation was made in 1996, and in July 1996, the Redwine site was officially designated an SAL by the Texas Historical Commission, the first SAL in Smith County. This paper describes our findings, and discusses the artifacts and plant and animal remains recovered during the work. We also provide information on the 1960s excavations of a small house mound at the site, along with the grave goods recovered by Sam Whiteside from the four Redwine site burials

Gamma radiation survey of the LDEF spacecraft

The retrieval of the Long Duration Exposure Facility spacecraft in January 1990 after nearly six years in orbit offered a unique opportunity to study the long term buildup of induced radioactivity in the variety of materials on board. We conducted the first complete gamma-ray survey of a large spacecraft on LDEF shortly after its return to earth. A surprising observation was the Be-7 activity which was seen primarily on the leading edge of the satellite, implying that it was picked up by LDEF in orbit. This is the first known evidence for accretion of a radioactive isotope onto an orbiting spacecraft. Other isotopes observed during the survey, the strongest being Na-22, are all attributed to activation of spacecraft components. Be-7 is a spallation product of cosmic rays on nitrogen and oxygen in the upper atmosphere. However, the observed density is much greater than expected due to cosmic-ray production in situ. This implies transport of Be-7 from much lower altitudes up to the LDEF orbit

Kinks in dipole chains

It is shown that the topological discrete sine-Gordon system introduced by Speight and Ward models the dynamics of an infinite uniform chain of electric dipoles constrained to rotate in a plane containing the chain. Such a chain admits a novel type of static kink solution which may occupy any position relative to the spatial lattice and experiences no Peierls-Nabarro barrier. Consequently the dynamics of a single kink is highly continuum like, despite the strongly discrete nature of the model. Static multikinks and kink-antikink pairs are constructed, and it is shown that all such static solutions are unstable. Exact propagating kinks are sought numerically using the pseudo-spectral method, but it is found that none exist, except, perhaps, at very low speed.Comment: Published version. 21 pages, 5 figures. Section 3 completely re-written. Conclusions unchange