Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Using DNA as a drug : challenges and opportunities for process engineers

The maturing of the biotechnology industry and a focus on productivity has seen a shift from discovery science to small-scale bench-top research to higher productivity, large scale production. Health companies are aggressively expanding their biopharmaceutical interests, an expansion which is facilitated by biochemical and bioprocess engineering. An area of continuous growth is vaccines. Vaccination will be a key intervention in the case of an influenza pandemic. The global manufacturing capacity for fast turn around vaccines is currently woefully inadequate at around 300 million shots. As the prevention of epidemics requires > 80 % vaccination, in theory the world should currently be aiming for the ability to produce around 5.3 billion vaccines. Presented is a production method for the creation of a fast turn around DNA vaccine. A DNA vaccine could have a production time scale of as little as two weeks. This process has been harnessed into a pilot scale production system for the creation of a pre-clinical grade malaria vaccine in a collaborative project with the Coppel Lab, Department of Microbiology, Monash University. In particular, improvements to the fermentation, chromatography and delivery stages will be discussed. Consideration will then be given as to how the fermentation stage affects the mid and downstream processing stages.

Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders. In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed. In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.

Computational analysis of epigenetic information in human DNA sequences

Over the last few years, investigations of human epigenetic profiles have identified key elements of change to be Histone Modifications, stable and heritable DNA methylation and Chromatin remodeling. These factors determine gene expression levels and characterise conditions leading to disease. In order to extract information embedded in long DNA sequences, data mining and pattern recognition tools are widely used, but efforts have been limited to date with respect to analyzing epigenetic changes, and their role as catalysts in disease onset. Useful insight, however, can be gained by investigation of associated dinucleotide distributions. The focus of this paper is to explore specific dinucleotides frequencies across defined regions within the human genome, and to identify new patterns between epigenetic mechanisms and DNA content. Signal processing methods, including Fourier and Wavelet Transformations, are employed and principal results are reported.

DNA molecules and future blockbuster therapeutics

Deoxyribonucleic acid molecules are heralding a new generation of reverse - engineered biopharmaceuticals. In terms of potential application in gene medicine, plasmid DNA (pDNA) vectors have exceptional therapeutic and immunological profiles as they are free from safety concerns associated with viral vectors, display non-toxicity and are simpler to develop. This presentation will discuss the potential applications of pDNA molecules in vaccine development and gene therapy, pilot-scale production of pDNA-based biopharmaceuticals and the controlled delivery of therapeutic sequences in biodegradable polymers to different target cells via the nasal route.

Biomechanics of transfemoral amputees fitted with osseointegrated fixation: Loading data for evidence-based practice

This presentation will provide an overview of the load applied on the residuum of transfemoral amputees fitted with an osseointegrated fixation during (A) rehabilitation, including static and dynamic load bearing exercises (e.g., rowing, adduction, abduction, squat, cycling, walking with aids), and (B) activities of daily living including standardized activities (e.g., level walking in straight line and around a circle, ascending and descending slopes and stairs) and activities in real world environments.

Review of direct measurement of inner prosthesis loading in transfemoral amputation: Potential benefits for evidence-based practice

The understanding of the load applied on the residuum through the prosthesis of individuals with transfemoral amputation (TFA) is essential to address a number of concerns that could strongly reduce their quality of life (e.g., residuum skin lesion, prosthesis fitting, alignment). This inner prosthesis loading could be estimated using a typical gait laboratory relying on inverse dynamics equations. Alternative, technological advances proposed over the last decade enabled direct measurement of this kinetic information in a broad variety of situations that could potentially be more relevant in clinical settings. The purposes of this presentation are (A) to review the literature about recent developments in measure and analyses of inner prosthesis loading of TFA, and (B) to extract information that could potentially contribute to a better evidence-based practice.

Direct measurement of prosthetic loading for evidence-based practice

The demand for an evidence-based clinical practice involving lower limb amputees is increasing. Some of the critical care decisions are related to the loading applied on the residuum partially responsible for comfort and functional outcome. This loading can be assessed using inverse dynamics equations. Typically, this method requires a gait laboratory (e.g., 3D motion analysis system, force-plates). It is mainly suited for the analysis only few steps of walking while being expensive and labour intensive. However, recent scientific and industrial developments demonstrated that discrete and light portable sensors can be placed within the prosthesis to measure accurately the loading during an unlimited number of steps and activities of daily living. Several studies indicated that method based on direct measurements might provide more realistic results. Furthermore, it is a user-friendly method more accessible to clinicians, such as prosthetists. The purpose of this symposium will be to give an overview of these additional opportunities for clinicians to obtain relevant data for evidence-based practice. The three main aims will be: • To present some of the equipment used for direct measurements, • To propose ways to analyse some key data sets, • To give some practical example of data sets for transtibial and transfemoral amputees.

Mechanisms of cisplatin resistance: DNA repair and cellular implications

Cisplatin (cis-diamminedichloroplatinum (II)), is a platinum based chemotherapeutic employed in the clinic to treat patients with lung, ovarian, colorectal or head and neck cancers. Cisplatin acts to induce tumor cell death via multiple mechanisms. The best characterized mode of action is through irreversible DNA cross-links which activate DNA damage signals leading to cell death via the intrinsic mitochondrial apoptosis pathway. However, the primary issue with cisplatin is that while patients initially respond favorably, sustained cisplatin therapy often yields chemoresistance resulting in therapeutic failure. In this chapter, we review the DNA damage and repair pathways that contribute to cisplatin resistance. We also examine the cellular implications of cisplatin resistance that may lead to selection of subpopulations of cells within a tumor. In better understanding the mechanisms conferring cisplatin resistance, novel targets may be identified to restore drug sensitivity.

BRCA1 deficiency exacerbates estrogen-induced DNA damage and genomic instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell cycle arrest and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here we report that estrogen and estrogen metabolites can cause DNA double strand breaks (DSB) in estrogen receptor-α negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability. We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolising enzymes, such as CYP1A1, in breast cells. Lastly, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumours in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types.

The role of DNA repair pathways in cisplatin resistant lung cancer

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer.

Identification of a BRCA1-mRNA splicing complex required for efficient DNA repair and maintenance of genomic stability

Mutations within BRCA1 predispose carriers to a high risk of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through the assembly of multiple protein complexes involved in DNA repair, cell-cycle arrest, and transcriptional regulation. Here, we report the identification of a DNA damage-induced BRCA1 protein complex containing BCLAF1 and other key components of the mRNA-splicing machinery. In response to DNA damage, this complex regulates pre-mRNA splicing of a number of genes involved in DNA damage signaling and repair, thereby promoting the stability of these transcripts/proteins. Further, we show that abrogation of this complex results in sensitivity to DNA damage, defective DNA repair, and genomic instability. Interestingly, mutations in a number of proteins found within this complex have been identified in numerous cancer types. These data suggest that regulation of splicing by the BRCA1-mRNA splicing complex plays an important role in the cellular response to DNA damage.