Determination of urinary thymidine glycol using affinity chromatography, HPLC and post-column reaction detection : a biomarker of oxidative DNA damage upon kidney transplantation

Reactive oxygen species are generated during ischaemia-reperfusion of tissue. Oxidation of thymidine by hydroxyl radicals (HO) leads to the formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol is excreted in urine and can be used as biomarker of oxidative DNA damage. Time dependent changes in urinary excretion rates of thymidine glycol were determined in six patients after kidney transplantation and in six healthy controls. A new analytical method was developed involving affinity chromatography and subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) with a post-column chemical reaction detector and endpoint fluorescence detection. The detection limit of this fluorimetric assay was 1.6 ng thymidine glycol per ml urine, which corresponds to about half of the physiological excretion level in healthy control persons. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum around 48 h. The excretion rate remained elevated until the end of the observation period of 10 days. Severe proteinuria with an excretion rate of up to 7.2 g of total protein per mmol creatinine was also observed immediately after transplantation and declined within the first 24 h of allograft function (0.35 + 0.26 g/mmol creatinine). The protein excretion pattern, based on separation of urinary proteins on sodium dodecyl sulphate-polyacrylamide gel electrophorosis (SDS-PAGE), as well as excretion of individual biomarker proteins, indicated nonselective glomerular and tubular damage. The increased excretion of thymidine glycol after kidney transplantation may be explained by ischaemia-reperfusion induced oxidative DNA damage of the transplanted kidney.

DNA binding study of isophorone in rats and mice

Following isophorone exposure, in a 2-year study with F344 rats and B6C3F1 mice performed under the National Toxicology Program (NTP), an elevated incidence of tumors was observed in male rats (kidney tumors) and male mice (liver tumors). Female rats and mice showed no elevation of tumor rates by isophorone (NTP 1986).

A/Effective connections : mobility, technology and well-being

This article investigates the significance of internet communication technologies for mediating affect in ways that help promote feelings of well-being among recently arrived migrants from culturally and linguistically diverse communities (CALD) in Australia. It is based on a qualitative study that focuses on the internet's communicative capabilities for maintaining kinship ties in homeland countries, and for forging new connections in the host city of Brisbane during the early re-settlement period. Through the experience of ‘presence’ and affective communities, it emphasizes the ways in which visually mediated interaction helps to combat feelings of social isolation and loneliness. The study finds that internet use is creating new forms of sociality among migrants and plays a key role in the re-settlement period. It highlights the importance of publicly available computers and training for migrants.

DNA methylation at the novel CpG sites in the promoter of MED15/PCQAP gene as a biomarker for head and neck cancers

Head and neck cancers (HNCs) represent a significant and ever-growing burden to the modern society, mainly due to the lack of early diagnostic methods. A significant number of HNCs is often associated with drinking, smoking, chewing beetle nut, and human papilloma virus (HPV) infections. We have analyzed DNA methylation patterns in tumor and normal tissue samples collected from head and neck squamous cell carcinoma (HNSCC) patients who were smokers. We have identified novel methylation sites in the promoter of the mediator complex subunit 15 (MED15/PCQAP) gene (encoing a co-factor important for regulation of transcription initiation for promoters of many genes), hypermethylated specifically in tumor cells. Two clusters of CpG dinucleotides methylated in tumors, but not in normal tissue from the same patients, were identified. These CpG methylation events in saliva samples were further validated in a separate cohort of HNSCC patients (who developed cancer due to smoking or HPV infections) and healthy controls using methylation-specific PCR (MSP). We used saliva as a biological medium because of its non-invasive nature, close proximity to the tumors, easiness and it is an economically viable option for large-scale screening studies. The methylation levels for the two identified CpG clusters were significantly different between the saliva samples collected from healthy controls and HNSCC individuals (Welch's t-test returning P, 0.05 and Mann-Whitney test P, 0.01 for both). The developed MSP assays also provided a good discriminative ability with AUC values of 0.70 (P, 0.01) and 0.63 (P, 0.05). The identified novel CpG methylation sites may serve as potential non-invasive biomarkers for detecting HNSCC. © the authors.

Methods for extracting genomic DNA from whole blood samples : current perspectives

Deoxyribonucleic acid (DNA) extraction has considerably evolved since it was initially performed back in 1869. It is the first step required for many of the available downstream applications used in the field of molecular biology. Whole blood samples are one of the main sources used to obtain DNA, and there are many different protocols available to perform nucleic acid extraction on such samples. These methods vary from very basic manual protocols to more sophisticated methods included in automated DNA extraction protocols. Based on the wide range of available options, it would be ideal to determine the ones that perform best in terms of cost-effectiveness and time efficiency. We have reviewed DNA extraction history and the most commonly used methods for DNA extraction from whole blood samples, highlighting their individual advantages and disadvantages. We also searched current scientific literature to find studies comparing different nucleic acid extraction methods, to determine the best available choice. Based on our research, we have determined that there is not enough scientific evidence to support one particular DNA extraction method from whole blood samples. Choosing a suitable method is still a process that requires consideration of many different factors, and more research is needed to validate choices made at facilities around the world.

Day in the Lab : Urban Informatics Research Lab, Queensland University of Technology

The Urban Informatics Research Lab brings together a group of people who focus their research on interdisciplinary topics at the intersection of social, spatial, and technical research domains—that is, people, place, and technology. Those topics are spread across the breadth of urban life—its contemporary issues and its needs, as well as the design opportunities that we have as individuals, groups, communities, and as a whole society. The lab’s current research areas include urban planning and design, civic innovation, mobility and transportation, education and connected learning, environmental sustainability, and food and urban agriculture. The common denominator of the lab’s approach is user-centered design research directed toward understanding, conceptualizing, developing, and evaluating sociotechnical practices as well as the opportunities afforded by innovative digital technology in urban environments.

Detection of DNA mutations using novel SERS (Surface-Enhanced Raman Spectroscopy) diagnostic platform

This article describes the detection of DNA mutations using novel Au-Ag coated GaN substrate as SERS (surface-enhanced Raman spectroscopy) diagnostic platform. Oligonucleotide sequences corresponding to the BCR-ABL (breakpoint cluster region-Abelson) gene responsible for development of chronic myelogenous leukemia were used as a model system to demonstrate the discrimination between the wild type and Met244Val mutations. The thiolated ssDNA (single-strand DNA) was immobilized on the SERS-active surface and then hybridized to a labeled target sequence from solution. An intense SERS signal of the reporter molecule MGITC was detected from the complementary target due to formation of double helix. The SERS signal was either not observed, or decreased dramatically for a negative control sample consisting of labeled DNA that was not complementary to the DNA probe. The results indicate that our SERS substrate offers an opportunity for the development of novel diagnostic assays.

Law students' perspectives of the affect of academics' use of technology

Previous work within the Faculty of Law, QUT had considered law students perceptions and use of technology and how to manage that use without it becoming a distraction. Students willingness to use technology for their learning purposes, however, had not been tested. The research seeks to understand the affect of law academics in class use of technology for both law and justice students. Students use and their perception of academics use in lectures and tutorials was tested by means of an online survey conducted on an anonymous and voluntary basis. The analysis of results revealed that the majority of respondents rarely use technology in class for their learning purposes. However, most indicated that academic in class use of technology enabled their learning. The research also reinforced the need to make any level of engagement with technology meaningful for students. In particular it identified the need to ensure that students are enabled, by appropriate training, in their use of any required databases or software.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage

Purpose: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. Materials and methods: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (137Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. Results: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 μM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 μM. Conclusions: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Evaluation of the efficacy of radiation-modifying compounds using γH2AX as a molecular marker of DNA double-strand breaks

Radiation therapy is a widely used therapeutic approach for cancer. To improve the efficacy of radiotherapy there is an intense interest in combining this modality with two broad classes of compounds, radiosensitizers and radioprotectors. These either enhance tumour-killing efficacy or mitigate damage to surrounding non-malignant tissue, respectively. Radiation exposure often results in the formation of DNA double-strand breaks, which are marked by the induction of H2AX phosphorylation to generate γH2AX. In addition to its essential role in DDR signalling and coordination of double-strand break repair, the ability to visualize and quantitate γH2AX foci using immunofluorescence microscopy techniques enables it to be exploited as an indicator of therapeutic efficacy in a range of cell types and tissues. This review will explore the emerging applicability of γH2AX as a marker for monitoring the effectiveness of radiation-modifying compounds.

Methylproamine protects against ionizing radiation by preventing DNA double-strand breaks

Purpose The majority of cancer patients will receive radiotherapy (RT), therefore, investigations into advances of this modality are important. Conventional RT dose intensities are limited by adverse responses in normal tissues and a primary goal is to ameliorate adverse normal tissue effects. The aim of these experiments is to further our understanding regarding the mechanism of radioprotection by the DNA minor groove binder, methylproamine, in a cellular context at the DNA level. Materials and methods We used immunocytochemical methods to measure the accumulation of phosphorylated H2AX (γH2AX) foci following ionizing radiation (IR) in patient-derived lymphoblastoid cells exposed to methylproamine. Furthermore, we performed pulsed field gel electrophoresis DNA damage and repair assays to directly interrogate the action of methylproamine on DNA in irradiated cells. Results We found that methylproamine-treated cells had fewer γH2AX foci after IR compared to untreated cells. Also, the presence of methylproamine decreased the amount of lower molecular weight DNA entering the gel as shown by the pulsed field gel electrophoresis assay. Conclusions These results suggest that methylproamine acts by preventing the formation of DNA double-strand breaks (dsbs) and support the hypothesis that radioprotection by methylproamine is mediated, at least in part, by decreasing initial DNA damage.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Comparing the effectiveness of education and technology in reducing wood smoke pollution: A field experiment

This study describes a field experiment assessing the effectiveness of education and technological innovation in reducing air pollution generated by domestic wood heaters. Two-hundred and twenty four households from a small regional center in Australia were randomly assigned to one of four experimental conditions: (1) Education only – households received a wood smoke reduction education pack containing information about the negative health impacts of wood smoke pollution, and advice about wood heater operation and firewood management; (2) SmartBurn only – households received a SmartBurn canister designed to improve combustion and help wood fires burn more efficiently, (3) Education and SmartBurn, and (4) neither Education nor SmartBurn (control). Analysis of covariance, controlling for pre-intervention household wood smoke emissions, wood moisture content, and wood heater age, revealed that education and SmartBurn were both associated with significant reduction in wood smoke emissions during the post-intervention period. Follow-up mediation analyses indicated that education reduced emissions by improving wood heater operation practices, but not by increasing health risk perceptions. As predicted, SmartBurn exerted a direct effect on emission levels, unmediated by wood heater operation practices or health risk perceptions.

Non-destructive DNA extraction from Chironomidae, including of fragile pupal exuviae, extends analysable collections and enhances vouchering

It is well established that the traditional taxonomy and nomenclature of Chironomidae relies on adult males whose usually characteristic genitalia provide evidence of species distinction. In the early days some names were based on female adults of variable distinctiveness – but females are difficult to identify (Ekrem et al. 2010) and many of these names remain dubious. In Russia especially, a system based on larval morphology grew in parallel to the conventional adult-based system. The systems became reconciled with the studies that underlay the production of the Holarctic generic keys to Chironomidae, commencing notably with the larval volume (Wiederholm, 1983). Ever since Thienemann’s pioneering studies, it has been evident that the pupa, notably the cast skins (exuviae) provide a wealth of features that can aid in identification (e.g. Wiederholm, 1986). Furthermore, the pupae can be readily associated with name-bearing adults when a pharate (‘cloaked’) adult stage is visible within the pupa. Association of larvae with the name-bearing later stages has been much more difficult, time-consuming and fraught with risk of failure. Yet it is identification of the larval stage that is needed by most applied researchers due to the value of the immature stages of the family in aquatic monitoring for water quality, although the pupal stage also has advocates (reviewed by Sinclair & Gresens, 2008). Few use the adult stage for such purposes as their provenance and association with the water body can be verified only by emergence trapping, and sampling of adults lies outside regular aquatic monitoring protocols.