Physical, chemical, biological and ecotoxicological properties of wastewater discharged from Davis Station, Antarctica

The properties and toxicity of untreatedwastewater at Davis Station, East Antarctica,were investigated to inform decisions regarding the appropriate level of treatment for local discharge purposes and more generally, to better understand the risk associated with dispersal and impact of wastewaters in Antarctica. Suspended solids, nutrients (nitrogen, phosphorus), biological oxygen demand (BOD), metals, organic contaminants, surfactants and microbiological load were measured at various locations throughout the wastewater discharge system. Wastewater quality and properties varied greatly between buildings on station, each ofwhich has separate holding tanks. Nutrients, BOD and settleable solid levelswere higher than standard municipal wastewaters. Microbiological loads were typical of untreated wastewater. Contaminants detected in the wastewater included metals and persistent organic compounds, mainly polybrominated diphenyl ethers (PBDEs). The toxicity of wastewater was also investigated in laboratory bioassays using two local Antarctic marine invertebrates, the amphipod Paramoera walkeri and the microgastropod Skenella paludionoides. Animals were exposed to a range of wastewater concentrations from3% to 68% (test 1) or 63% (test 2) over 21 days with survival monitored daily. Significant mortality occurred in all concentrations of wastewater after 14 to 21 days, and at higher concentrations (50–68% wastewater) mortality occurred after only one day. Results indicate that the local receiving marine environment at Davis Station is at risk from existing wastewater discharges, and that advanced treatment is required both to remove contaminants shown to cause toxicity to biota, as well as to reduce the environmental risks associated with non-native micro-organisms in wastewater.

Treatment for gender dysphoria in children : the new legal, ethical and clinical landscape

- Gender dysphoria is a condition in which a child's subjectively felt identity and gender are not congruent with her or his biological sex. Because of this, the child suffers clinically significant distress or impairment in social functioning. - The Family Court of Australia has recently received an increasing number of applications seeking authorisation for the provision of hormones to treat gender dysphoria in children. - Some medical procedures and interventions performed on children are of such a grave nature that court authorisation must be obtained to render them lawful. These procedures are referred to as special medical procedures. - Hormonal therapy for the treatment of gender dysphoria in children is provided in two stages occurring years apart. Until recently, both stages of treatment were regarded by courts as special medical treatments, meaning court authorisation had to be provided for both stages. - In a significant recent development, courts have drawn a distinction between the two stages of treatment, permitting parents to consent to the first stage. In addition, it has been held that a child who is determined by a court to be Gillick competent can consent to stage 2 treatment. - The new legal developments concerning treatment for gender dysphoria are of ethical, clinical and practical importance to children and their families, and to medical practitioners treating children with gender dysphoria. Medical practitioners should benefit from an understanding of the recent developments in legal principles. This will ensure that they have up-to-date information about the circumstances under which treatment may be conducted with parental consent, and those in which they must seek court authorisation.

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.

Versatility of polymethacrylate monoliths for chromatographic purification of biomolecules

Polymethacrylate monoliths, specifically poly(glycidyl methacrylate-co-ethylene dimethacrylate) or poly(GMA-co-EDMA) monoliths, are a new generation of chromatographic supports and are significantly different from conventional particle-based adsorbents, membranes, and other monolithic supports for biomolecule purification. Similar to other monoliths, polymethacrylate monoliths possess large pores which allow convective flow of mobile phase and result in high flow rates at reduced pressure drop, unlike particulate supports. The simplicity of the adsorbent synthesis, pH resistance, and the ease and flexibility of tailoring their pore size to that of the target biomolecule are the key properties which differentiate polymethacrylate monoliths from other monoliths. Polymethacrylate monoliths are endowed with reactive epoxy groups for easy functionalization (with anion-exchange, hydrophobic, and affinity ligands) and high ligand retention. In this review, the structure and performance of polymethacrylate monoliths for chromatographic purification of biomolecules are evaluated and compared to those of other supports. The development and use of polymethacrylate monoliths for research applications have grown rapidly in recent times and have enabled the achievement of high through-put biomolecule purification on semi-preparative and preparative scales.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

A thermal expulsion approach to homogeneous large-volume methacrylate monolith preparation; enabling large-scale rapid purification of biomolecules

Numerous efforts have been dedicated to the synthesis of large-volume methacrylate monoliths for large-scale biomolecules purification but most were obstructed by the enormous release of exotherms during preparation, thereby introducing structural heterogeneity in the monolith pore system. A significant radial temperature gradient develops along the monolith thickness, reaching a terminal temperature that supersedes the maximum temperature required for structurally homogenous monoliths preparation. The enormous heat build-up is perceived to encompass the heat associated with initiator decomposition and the heat released from free radical-monomer and monomer-monomer interactions. The heat resulting from the initiator decomposition was expelled along with some gaseous fumes before commencing polymerization in a gradual addition fashion. Characteristics of 80 mL monolith prepared using this technique was compared with that of a similar monolith synthesized in a bulk polymerization mode. An extra similarity in the radial temperature profiles was observed for the monolith synthesized via the heat expulsion technique. A maximum radial temperature gradient of only 4.3°C was recorded at the center and 2.1°C at the monolith peripheral for the combined heat expulsion and gradual addition technique. The comparable radial temperature distributions obtained birthed identical pore size distributions at different radial points along the monolith thickness.

Large-volume methacrylate monolith for plasmid purification : process engineering approach to synthesis and application

The extent of exothermicity associated with the construction of large-volume methacrylate monolithic columns has somewhat obstructed the realisation of large-scale rapid biomolecule purification especially for plasmid-based products which have proven to herald future trends in biotechnology. A novel synthesis technique via a heat expulsion mechanism was employed to prepare a 40 mL methacrylate monolith with a homogeneous radial pore structure along its thickness. Radial temperature gradient was recorded to be only 1.8 °C. Maximum radial temperature recorded at the centre of the monolith was 62.3 °C, which was only 2.3 °C higher than the actual polymerisation temperature. Pore characterisation of the monolithic polymer showed unimodal pore size distributions at different radial positions with an identical modal pore size of 400 nm. Chromatographic characterisation of the polymer after functionalisation with amino groups displayed a persistent dynamic binding capacity of 15.5 mg of plasmid DNA/mL. The maximum pressure drop recorded was only 0.12 MPa at a flow rate of 10 mL/min. The polymer demonstrated rapid separation ability by fractionating Escherichia coli DH5α-pUC19 clarified lysate in only 3 min after loading. The plasmid sample collected after the fast purification process was tested to be a homogeneous supercoiled plasmid with DNA electrophoresis and restriction analysis.

Binding mechanism of affinity ligands for purification of plasmid DNA

Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity chromatography makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. In this study, surface plasmon resonance (SPR) based Biacore system has been employed for the detection and quantification of the binding between lac operon (lacO) sequence contained in a pDNA and synthetic peptides based on the DNA-binding domain of the lac repressor protein, lad. The equilibrium dissociation constant (K D) and association and dissociation rate constants (ka, kd) for the interaction between plasmid DNA and designed peptides were determined.

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

A monolithic stationary phase was prepared via free radical co-polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized. with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α-pUC19 culture in a ribonuclease/ protease-free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 min anion-exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr-AGE), enzyme restriction analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This non-toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level.

Plasmid DNA purification and formulation for vaccine applications

Plasmid DMA offers the promise of a new generation of pharmaceuticals that will address the often overlooked issue of vaccine production by offering a simple and reproducible method for producing a vaccine. Through reverse engineering, production could be reduced from up to 9 months to as little as 1 month. Simplified development and faster turn-around times means that DMA offers a solution to the vaccine crisis and will help to contain future viral outbreaks by enabling the production of a vaccine against new viral strains in the shortest possible time. Work currently being completed in the area of plasmid DMA production, purification and encapsulation will be presented.

Preparation, Analysis and Use of an Affinity Adsorbent for the Purification of GST Fusion Protein

Methods are presented for the preparation, ligand density analysis and use of an affinity adsorbent for the purification of a glutathione S-transferase (GST) fusion protein in packed and expanded bed chromatographic processes. The protein is composed of GST fused to a zinc finger transcription factor (ZnF). Glutathione, the affinity ligand for GST purification, is covalently immobilized to a solid-phase adsorbent (Streamline™). The GST–ZnF fusion protein displays a dissociation constant of 0.6 x10-6 M to glutathione immobilized to Streamline™. Ligand density optimization, fusion protein elution conditions (pH and glutathione concentration) and ligand orientation are briefly discussed.

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.

Materials, methods and systems for purification and/or seperation

Materials, methods and systems are provided for the purifn., filtration and​/or sepn. of certain mols. such as certain size biomols. Certain embodiments relate to supports contg. at least one polymethacrylate polymer engineered to have certain pore diams. and other properties, and which can be functionally adapted to for certain purifications, filtrations and​/or sepns. Biomols. are selected from a group consisting of: polynucleotide mols., oligonucleotide mols. including antisense oligonucleotide mols. such as antisense RNA and other oligonucleotide mols. that are inhibitory of gene function such as small interfering RNA (siRNA)​, polypeptides including proteinaceous infective agents such as prions, for example, the infectious agent for CJD, and infectious agents such as viruses and phage.