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.

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.

Nonprofit Service Delivery for Government : Accountability Relationships and Mechanisms

When government purchases social services under contract from a nonprofit organisation, a clear accountability relationship is created. The NPO must give an account for the use of the funds and achievement of outcomes to the funder. This paper explores how accountability is enacted in two different types of funding relationships in Queensland. Support is found for the argument that different relationships have different approaches to accountability.

Phylogenetics of small horseshoe bats from east Asia based on mitochondrial DNA sequence variation

We undertook analyses of mitochondrial DNA gene sequences and echolocation calls to resolve phylogenetic relationships among the related bat taxa Rhinolophus pusillus (sampled across China), R. monoceros (Taiwan), R. cornutus (main islands of Japan), and R. c. pumilus (Okinawa, Japan), Phylogenetic trees and genetic divergence analyses were constructed by combining new complete mitochondrial cytochrome-b gene sequences and partial mitochondrial control region sequences with published sequences. Our work showed that these 4 taxa formed monophyletic groups in the phylogenetic tree. However, low levels of sequence divergence among the taxa, together with similarities in body size and overlapping echolocation call frequencies, point to a lack of taxonomic distinctiveness. We therefore suggest that these taxa are better considered as geographical subspecies rather than distinct species, although this should not diminish the conservation importance of these island populations, which are important evolutionarily significant units. Based on our findings, we suggest that the similarities in body size and echolocation call frequency in these rhinolophids result from their recent common ancestry, whereas similarities in body size and call frequency with R. hipposideros of Europe are the result of convergent evolution.

Learnings, case studies and guidelines for establishing shared and collaborative service delivery in the non-government sector: Evaluation of the Multi-Tenant Service Centre (MTSC) Pilots Project

This report presents learnings, case studies, guidelines and resources for non-government organisations that are planning to implement shared or collaborative arrangements with other agencies. It summarises results from an evaluation of the implementation phase of the Multi-Tenant Service Centre (MTSC) Pilots Project, which was completed in June 2008. This evaluation shows that developing and implementing shared and collaborative arrangements is a complex process that presents many risks, challenges and barriers to success, but can have many potential benefits for non government organisations. As this report makes clear, there is no ‘one size fits all’ approach to this process. The MTSC Pilots Project was conducted by the Department of Communities (DoC), Queensland Government, as part of its Strengthening Non-Government Organisations strategy. The objective of the MTSC Pilots initiative was to co-locate separate service providers in an appropriately located centre, operating with effective and transparent management, which enabled service providers to improve client services. Three MTSC consortiums in Mackay, Caboolture and Toowoomba were selected as the pilots over a four year period from 2006 – 2010.

Mitochondrial genome acquisition restores respiratory function and tumorigenic potential of cancer cells without mitochondrial DNA

We report that tumor cells devoid of their mitochondrial genome (mtDNA) show delayed tumor growth and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory supercomplexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest a novel pathophysiological process for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

Background Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Results Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. Conclusions This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

The harnessing of peptide-monolith constructs for single step plasmid DNA purification

The availability of synthetic peptides has paved the way for their use in tailor-made interactions with biomolecules. In this study, a 16mer LacI-based peptide was used as an affinity ligand to examine the scale up feasibility for plasmid DNA purification. First, the peptide was designed and characterized for the affinity purification of lacO containing plasmid DNA, to be employed as a high affinity ligand for the potential capturing of plasmid DNA in a single unit operation. It was found there were no discernible interactions with a control plasmid that did not encode the lacO nucleotide sequence. The dissociation equilibrium constant of the binding between the 16mer peptide and target pUC19 was 5.0 ± 0.5 × 10-8 M as assessed by surface plasmon resonance. This selectivity and moderated affinity indicate that the 16mer is suitable for the adsorption and chromatographic purification of plasmid DNA. The suitability of this peptide was then evaluated using a chromatography system with the 16mer peptide immobilized to a customized monolith to purify plasmid DNA, obtaining preferential purification of supercoiled pUC19. The results demonstrate the applicability of peptide-monolith supports to scale up the purification process for plasmid DNA using designed ligands via a biomimetic approach.

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Peptides constructed from α-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to "biomimics" of the DNA binding domain of LacI. Equilibrium dissociation constants (K D), association (k a), and dissociation rates (k d) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 ± 1.3 × 10 -10 M, 7.2 ± 0.6 × 10 -10 M, 4.5 ± 0.5 × 10 -8 M, and 6.2 ± 0.9 × 10 -6 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operon-repressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

Protein loaded mesoporous silica spheres as a controlled delivery platform

Background The adsorption of bovine serum albumin (BSA) onto mesoporous silica spheres (MPS) synthesized from silica colloids was studied employing real time in situ measurements. The stabilities of the BSA at different pH values, their isoelectric points and zeta potentials were determined in order to probe the interactions between the protein and the mesoporous silica. Results The pore size of MPS was designed for protein, and this, coupled with an in depth understanding of the physico-chemical characteristics of the protein and MPS has yielded a better binding capacity and delivery profile. The adsorption isotherm at pH 4.2 fitted the Langmuir model and displayed the highest adsorption capacity (71.43 mg mL-1 MPS). Furthermore, the delivery rates of BSA from the MPS under physiological conditions were shown to be dependent on the ionic strength of the buffer and protein loading concentration. Conclusion Economics and scale-up considerations of mesoporous material synthesized via destabilization of colloids by electrolyte indicate the scaleability and commercial viability of this technology as a delivery platform for biopharmaceutical applications.