Development of a log-quadratic model to describe microbial inactivation, illustrated by thermal inactivation of Clostridium botulinum

In the commercial food industry, demonstration of microbiological safety and thermal process equivalence often involves a mathematical framework that assumes log-linear inactivation kinetics and invokes concepts of decimal reduction time (DT), z values, and accumulated lethality. However, many microbes, particularly spores, exhibit inactivation kinetics that are not log linear. This has led to alternative modeling approaches, such as the biphasic and Weibull models, that relax strong log-linear assumptions. Using a statistical framework, we developed a novel log-quadratic model, which approximates the biphasic and Weibull models and provides additional physiological interpretability. As a statistical linear model, the log-quadratic model is relatively simple to fit and straightforwardly provides confidence intervals for its fitted values. It allows a DT-like value to be derived, even from data that exhibit obvious "tailing." We also showed how existing models of non-log-linear microbial inactivation, such as the Weibull model, can fit into a statistical linear model framework that dramatically simplifies their solution. We applied the log-quadratic model to thermal inactivation data for the spore-forming bacterium Clostridium botulinum and evaluated its merits compared with those of popular previously described approaches. The log-quadratic model was used as the basis of a secondary model that can capture the dependence of microbial inactivation kinetics on temperature. This model, in turn, was linked to models of spore inactivation of Sapru et al. and Rodriguez et al. that posit different physiological states for spores within a population. We believe that the log-quadratic model provides a useful framework in which to test vitalistic and mechanistic hypotheses of inactivation by thermal and other processes. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Predicting flow and transport in highly heterogeneous alluvial aquifers

Successful prediction of groundwater flow and solute transport through highly heterogeneous aquifers has remained elusive due to the limitations of methods to characterize hydraulic conductivity (K) and generate realistic stochastic fields from such data. As a result, many studies have suggested that the classical advective-dispersive equation (ADE) cannot reproduce such transport behavior. Here we demonstrate that when high-resolution K data are used with a fractal stochastic method that produces K fields with adequate connectivity, the classical ADE can accurately predict solute transport at the macrodispersion experiment site in Mississippi. This development provides great promise to accurately predict contaminant plume migration, design more effective remediation schemes, and reduce environmental risks. Key Points Non-Gaussian transport behavior at the MADE site is unraveledADE can reproduce tracer transport in heterogeneous aquifers with no calibrationNew fractal method generates heterogeneous K fields with adequate connectivity

Model-based analysis and optimization of bioreactor for hematopoietic stem cell cultivation

One of the problems to be solved in attaining the full potentials of hematopoietic stem cell (HSC) applications is the limited availability of the cells. Growing HSCs in a bioreactor offers an alternative solution to this problem. Besides, it also offers the advantages of eliminating labour intensive process as well as the possible contamination involved in the periodic nutrient replenishments in the traditional T-flask stem cell cultivation. In spite of this, the optimization of HSC cultivation in a bioreactor has been barely explored. This manuscript discusses the development of a mathematical model to describe the dynamics in nutrient distribution and cell concentration of an ex vivo HSC cultivation in a microchannel perfusion bioreactor. The model was further used to optimize the cultivation by proposing three alternative feeding strategies in order to prevent the occurrence of nutrient limitation in the bioreactor. The evaluation of these strategies, the periodic step change increase in the inlet oxygen concentration, the periodic step change increase in the media inflow, and the feedback control of media inflow, shows that these strategies can successfully improve the cell yield of the bioreactor. In general, the developed model is useful for the design and optimization of bioreactor operation.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

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.

Enhancing methacrylate-monolith-based downstream processes to champion plasmid DNA production

Increasing numbers of preclinical and clinical studies are utilizing pDNA (plasmid DNA) as the vector. In addition, there has been a growing trend towards larger and larger doses of pDNA utilized in human trials. The growing demand on pDNA manufacture leads to pressure to make more in less time. A key intervention has been the use of monoliths as stationary phases in liquid chromatography. Monolithic stationary phases offer fast separation to pDNA owing to their large pore size, making pDNA in the size range from 100 nm to over 300 nm easily accessible. However, the convective transport mechanism of monoliths does not guarantee plasmid purity. The recovery of pure pDNA hinges on a proper balance in the properties of the adsorbent phase, the mobile phase and the feedstock. The effects of pH and ionic strength of binding buffer, temperature of feedstock, active group density and the pore size of the stationary phase were considered as avenues to improve the recovery and purity of pDNA using a methacrylate-based monolithic adsorbent and Escherichia coli DH5α-pUC19 clarified lysate as feedstock. pDNA recovery was found to be critically dependent on the pH and ionic strength of the mobile phase. Up to a maximum of approx. 92% recovery was obtained under optimum conditions of pH and ionic strength. Increasing the feedstock temperature to 80°C increased the purity of pDNA owing to the extra thermal stability associated with pDNA over contaminants such as proteins. Results from toxicological studies of the plasmid samples using endotoxin standard (E. coli 0.55:B5 lipopolysaccharide) show that endotoxin level decreases with increasing salt concentration. It was obvious that large quantities of pure pDNA can be obtained with minimal extra effort simply by optimizing process parameters and conditions for pDNA purification.

Process engineering aspects of plasmid-based biopharmaceuticals production : tackling the threatening vaccine shortages to prevent global pandemics

Infectious diseases such as SARS, influenza and bird flu have the potential to cause global pandemics; a key intervention will be vaccination. Hence, it is imperative to have in place the capacity to create vaccines against new diseases in the shortest time possible. In 2004, The Institute of Medicine asserted that the world is tottering on the verge of a colossal influenza outbreak. The institute stated that, inadequate production system for influenza vaccines is a major obstruction in the preparation towards influenza outbreaks. Because of production issues, the vaccine industry is facing financial and technological bottlenecks: In October 2004, the FDA was caught off guard by the shortage of flu vaccine, caused by a contamination in a US-based plant (Chiron Corporation), one of the only two suppliers of US flu vaccine. Due to difficulties in production and long processing times, the bulk of the world's vaccine production comes from very small number of companies compared to the number of companies producing drugs. Conventional vaccines are made of attenuated or modified forms of viruses. Relatively high and continuous doses are administered when a non-viable vaccine is used and the overall protective immunity obtained is ephemeral. The safety concerns of viral vaccines have propelled interest in creating a viable replacement that would be more effective and safer to use.

Variation in parental magmas of Mt Rouse, a complex polymagmatic monogenetic volcano in the basaltic intraplate Newer Volcanics Province, southeast Australia

Monogenetic volcanoes have long been regarded as simple in nature, involving single magma batches and uncomplicated evolutions; however, recent detailed research into individual centres is challenging that assumption. Mt Rouse (Kolor) is the volumetrically largest volcano in the monogenetic Newer Volcanics Province of southeast Australia. This study presents new major, trace and Sr–Nd–Pb isotope data for samples selected on the basis of a detailed stratigraphic framework analysis of the volcanic products from Mt Rouse. The volcano is the product of three magma batches geochemically similar to Ocean–Island basalts, featuring increasing LREE enrichment with each magma batch (batches A, B and C) but no evidence of crustal contamination; the Sr–Nd–Pb isotopes define two groupings. Modelling suggests that the magmas were sourced from a zone of partial melting crossing the lithosphere–asthenosphere boundary, with batch A forming a large volume partial melt in the deep lithosphere (1.7 GPa/55.5 km); and batches B and C from similar areas within the shallow asthenosphere (1.88 GPa/61 km and 1.94 GPa/63 km, respectively). The formation and extraction of these magmas may have been due to high deformation rates in the mantle caused by edge-driven convection and asthenospheric upwelling. The lithosphere– asthenosphere boundary is important with respect to NVP volcanism. An eruption chronology involves sequential eruption of magma batches A, C and B, followed by simultaneous eruption of batches A and B. Mt Rouse is a complex polymagmatic monogenetic volcano that illustrates the complexity of monogenetic volcanism and demonstrates the importance of combining detailed stratigraphic analysis alongside systematic geochemical sampling.

Endotoxin removal : a critical step in vaccine production

A major drawback to the immunological potency of conventional vaccines, resulting in reduced level of immune responses, tissue injury, shock and high cytotoxicity, thus making their applications contraindicated in immunodeficiency diseases, is the presence of high contaminant concentrations in vaccine titers. Vaccine contamination arises from the simultaneous occurrence of competitive pathways resulting in the formation of other bio-products during cellular metabolism aside the pathways necessary for the production of vaccine molecules. One of such vaccine contaminating molecules is endotoxins which are mainly lipopolysaccharides (LPS) complexes found in the membrane of bacterial cell wall. The structural dynamics of these molecules make their removal from vaccine titers problematic, thus making vaccine endotoxin removal a major research endeavour. This presentation will discuss a novel technique for reducing the endotoxin level of vaccines. The technique commences with the disentanglement of endotoxin-vaccine molecular bonding and then capturing the vaccine molecules on an affinity monolith to separate the vaccine molecules from the endotoxins.

Sustainability of public health interventions to reduce the risk of dioxin exposure at severe dioxin hot spots in Vietnam

Severe dioxin contamination at Bien Hoa and Da Nang airbases, Vietnam is of international concern. Public Health risk reduction programs were implemented in Bien Hoa in 2007-2009 and in Da Nang in 2009-2011. In 2009 and 2011 we reported the encouraging results of these interventions in improving the knowledge, attitude and practices (KAP) of local residents in reducing the dioxin exposure risk through foods. In 2013 we revisited these dioxin hot spots, aimed to evaluate whether the results of the intervention were maintained and to identify factors affecting the sustainability of the programs. To assess this, 16 in-depth interviews, six focus group discussions, and pre and post intervention KAP surveys were undertaken. 800 respondents from six intervention wards and 200 respondents from Buu Long Ward (the control site) were randomly selected to participate in the surveys. The results showed that as of 2013, the programs were rated as "moderately sustained" with a score of 3.3 out of 5.0 (cut off points 2.5 to <3.5) for Bien Hoa, and "well sustained" with a score of 3.8 out of 5.0 (cut off points 3.5 to <4.5) for Da Nang. Most formal intervention program activities had ceased and dioxin risk communication activities were no longer integrated into local routine health education programs. However, the main outcomes were maintained and were better than that in the control ward. Migration, lack of official guidance from City People's Committees and local authorities as well as the politically sensitive nature of dioxin issues were the main challenges for the sustainability of the programs.

Inside the ‘Hurt Locker’: The combined effects of explosive ordnance disposal and chemical protective clothing on physiological tolerance time in extreme environments

Background Explosive ordnance disposal (EOD) technicians are often required to wear specialised clothing combinations that not only protect against the risk of explosion but also potential chemical contamination. This heavy (>35kg) and encapsulating ensemble is likely to increase physiological strain by increasing metabolic heat production and impairing heat dissipation. This study investigated the physiological tolerance times of two different chemical protective undergarments, commonly worn with EOD personal protective clothing, in a range of simulated environmental extremes and work intensities Methods Seven males performed eighteen trials wearing two ensembles. The trials involved walking on a treadmill at 2.5, 4 and 5.5 km.h-1 at each of the following environmental conditions, 21, 30 and 37°C wet bulb globe temperature (WBGT). The trials were ceased if the participants’ core temperature reached 39°C, if heart rate exceeded 90% of maximum, if walking time reached 60 minutes or due to volitional fatigue. Results Physiological tolerance times ranged from 8 to 60 min and the duration (mean difference: 2.78 min, P>0.05) were similar in both ensembles. A significant effect for environment (21>30>37°C WBGT, P<0.05) and work intensity (2.5>4>5.5 km.h-1, P< 0.05) was observed in tolerance time. The majority of trials across both ensembles (101/126; 80.1%) were terminated due to participants achieving a heart rate equivalent to greater than 90% of their maximum. Conclusions Physiological tolerance times wearing these two chemical protective undergarments, worn underneath EOD personal protective clothing, were similar and predominantly limited by cardiovascular strain.

Development of a hybrid pollution index for heavy metals in marine and estuarine sediments

Heavy metal pollution of sediments is a growing concern in most parts of the world, and numerous studies focussed on identifying contaminated sediments by using a range of digestion methods and pollution indices to estimate sediment contamination have been described in the literature. The current work provides a critical review of the more commonly used sediment digestion methods and identifies that weak acid digestion is more likely to provide guidance on elements that are likely to be bioavailable than other traditional methods of digestion. This work also reviews common pollution indices and identifies the Nemerow Pollution Index as the most appropriate method for establishing overall sediment quality. Consequently, a modified Pollution Index that can lead to a more reliable understanding of whole sediment quality is proposed. This modified pollution index is then tested against a number of existing studies and demonstrated to give a reliable and rapid estimate of sediment contamination and quality.

An Australian native plant: A novel antimicrobial for the treatment of MRSA

Chronic wounds, often associated with venous and arterial ulcers, diabetes and pressure sores, is an area of great concern. In Australia, the cost of treating chronic wounds is conservatively estimated at $285 million/annum for the treatment of pressure ulcers and $654 million annually for the treatment and management of leg ulcers. Current figures indicate that more than seven million people suffer from chronic wounds worldwide with Australians accounting for approximately 600,000 of this number. Bacterial infection of the wound site is a major issue as contamination of a chronic wound with methicillin-resistant Staphylococcus aureus (MRSA) significantly delays wound healing. Further, once systemic, current antibiotic therapies capable of treating the infection are limited. Aboriginal bush medicine has been used for thousands of years for the treatment of wounds and sores. Hence, we selected a native Australian plant to evaluate its bactericidal activity against MRSA.

Airborne culturable fungi in naturally ventilated primary school environments in a subtropical climate

There is currently a lack of reference values for indoor air fungal concentrations to allow for the interpretation of measurement results in subtropical school settings. Analysis of the results of this work established that, in the majority of properly maintained subtropical school buildings, without any major affecting events such as floods or visible mould or moisture contamination, indoor culturable fungi levels were driven by outdoor concentration. The results also allowed us to benchmark the “baseline range” concentrations for total culturable fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings. The measured concentration of total culturable fungi and three individual fungal genera were estimated using Bayesian hierarchical modelling. Pooling of these estimates provided a predictive distribution for concentrations at an unobserved school. The results indicated that “baseline” indoor concentration levels for indoor total fungi, Penicillium spp., Cladosporium spp. and Aspergillus spp. in such school settings were generally ≤ 1450, ≤ 680, ≤ 480 and ≤ 90 cfu/m3, respectively, and elevated levels would indicate mould damage in building structures. The indoor/outdoor ratio for most classrooms had 95% credible intervals containing 1, indicating that fungi concentrations are generally the same indoors and outdoors at each school. Bayesian fixed effects regression modeling showed that increasing both temperature and humidity resulted in higher levels of fungi concentration.

Decompression melting driving intraplate volcanism in Australia: Evidence from magnetotelluric sounding

A long-period magnetotelluric (MT) survey, with 39 sites covering an area of 270 by 150 km, has identified melt within the thinned lithosphere of Pleistocene-Holocene Newer Volcanics Province (NVP) in southeast Australia, which has been variously attributed to mantle plume activity or edge-driven mantle convection. Two-dimensional inversions from the MT array imaged a low-resistivity anomaly (10-30Ωm) beneath the NVP at ∼40-80 km depth, which is consistent with the presence of ∼1.5-4% partial melt in the lithosphere, but inconsistent with elevated iron content, metasomatism products or a hot spot. The conductive zone is located within thin juvenile oceanic mantle lithosphere, which was accreted onto thicker Proterozoic continental mantle lithosphere. We propose that the NVP owes its origin to decompression melting within the asthenosphere, promoted by lithospheric thickness variations in conjunction with rapid shear, where asthenospheric material is drawn by shear flow at a "step" at the base of the lithosphere.