Methods for isolation and cultivation of filamentous fungi

Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology.

Enzyme activities and biotechnological applications of cold-active microfungi

Fungi are eukaryotic organisms and considered to be less adaptable to extreme environments when compared to bacteria. While there are no thermophilic microfungi in a strict sense, some fungi have adapted to life in the cold. Cold-active microfungi have been isolated from the Antarctic and their enzyme activities explored with a view to finding new candidates for industrial use. On another front, environmental pollution by petroleum products in the Antarctic has led to a search for, and the subsequent discovery of, fungal isolates capable of degrading hydrocarbons. The work has paved the way to developing a bioremedial approach to containing this type of contamination in cold climates. Here we discuss our efforts to map the capability of Antarctic microfungi to degrade oil and also introduce a novel cold-active fungal lipase enzyme.

Ecological risk analysis of transgenic plants

Public concern about the safety of many forms of industrial technology are known to be linked to a range of factors including a perceived lack of confidence in regulatory decision making.1 The use of transgenic plants in agriculture may be seen as an issue that could generate similar concern. Criticism has been made about the completeness of knowledge on the potential for aberrant behaviour of genetically manipulated organisms (GMO's) in release environments, and the adequacy of existing pre­‐release screening and assessment methodologies (Goldberg & Tjaden, 1990). Such comments are important because any perceived shortcomings in the pre-release assessment of GMO safety may lead to decreased public support of the technology -­‐and the industry itself...

An investigation of Staphylococcus aureus and related species from flood affected and other environmental sources

This research investigated the microbial air quality of flooded houses in Brisbane suburbs following the January 2011 flood event. Flood waters can carry and spread human pathogenic bacteria, and these organisms can be dispersed into residential air by aerosolisation. This study found that the bacterial load was significantly different for indoor and outdoor areas of flood affected houses, but no significant differences were observed between flooded and non-flooded houses. This could be due to the rapid clean-up of flooded houses following the event. Molecular methods were used to identify and characterise staphylococcal species in residential air of flooded and non-flooded houses. A major finding was the diverse population of airborne staphylococci as well as the high rate of methicillin-resistance in these strains. By determining the genetic relatedness of residential air sourced staphylococci, a potential source for pathogenic strains can be identified.

The use of phospholipid fatty acid analysis to measure impact of acid rock drainage on microbial communities in sediments

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0-0.1 km), compared with sites further downstream (2.5-18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments' metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:2ω6; the sulfate-reducing bacteria marker 10Me16:1ω7; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.

Impact of a change in tillage and crop residue management practice on soil chemical and microbiological properties in a cereal-producing red duplex soil in NSW, Australia

The effect of a change of tillage and crop residue management practice on the chemical and micro-biological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0-5 cm of soil, in organic C, total N, electrical conductivity, NH4-N, NO3-N, soil moisture holding capacity, microbial biomass and CO2 respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0-5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.

Interaction between Arabidopsis Brca2 and its partners Rad51, Dmc1, and Dss1

The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).

Changes in healthcare-associated Staphylococcus aureus bloodstream infections after the introduction of a national hand hygiene initiative

Background. Interventions that prevent healthcare-associated infection should lead to fewer deaths and shorter hospital stays. Cleaning hands (with soap or alcohol) is an effective way to prevent the transmission of organisms, but rates of compliance with hand hygiene are sometimes disappointingly low. The National Hand Hygiene Initiative in Australia aimed to improve hand hygiene compliance among healthcare workers, with the goal of reducing rates of healthcare-associated infection. Methods. We examined whether the introduction of the National Hand Hygiene Initiative was associated with a change in infection rates. Monthly infection rates for healthcare-associated Staphylococcus aureus bloodstream infections were examined in 38 Australian hospitals across 6 states. We used Poisson regression and examined 12 possible patterns of change, with the best fitting pattern chosen using the Akaike information criterion. Monthly bed-days were included to control for increased hospital use over time. Results. The National Hand Hygiene Initiative was associated with a reduction in infection rates in 4 of the 6 states studied. Two states showed an immediate reduction in rates of 17% and 28%, 2 states showed a linear decrease in rates of 8% and 11% per year, and 2 showed no change in infection rates. Conclusions. The intervention was associated with reduced infection rates in most states. The failure in 2 states may have been because those states already had effective initiatives before the national initiative’s introduction or because infection rates were already low and could not be further reduced.

Bridging experiments, models and simulations : an integrative approach to validation in computational cardiac electrophysiology

Computational models in physiology often integrate functional and structural information from a large range of spatio-temporal scales from the ionic to the whole organ level. Their sophistication raises both expectations and scepticism concerning how computational methods can improve our understanding of living organisms and also how they can reduce, replace and refine animal experiments. A fundamental requirement to fulfil these expectations and achieve the full potential of computational physiology is a clear understanding of what models represent and how they can be validated. The present study aims at informing strategies for validation by elucidating the complex interrelations between experiments, models and simulations in cardiac electrophysiology. We describe the processes, data and knowledge involved in the construction of whole ventricular multiscale models of cardiac electrophysiology. Our analysis reveals that models, simulations, and experiments are intertwined, in an assemblage that is a system itself, namely the model-simulation-experiment (MSE) system. Validation must therefore take into account the complex interplay between models, simulations and experiments. Key points for developing strategies for validation are: 1) understanding sources of bio-variability is crucial to the comparison between simulation and experimental results; 2) robustness of techniques and tools is a pre-requisite to conducting physiological investigations using the MSE system; 3) definition and adoption of standards facilitates interoperability of experiments, models and simulations; 4) physiological validation must be understood as an iterative process that defines the specific aspects of electrophysiology the MSE system targets, and is driven by advancements in experimental and computational methods and the combination of both.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Structure and function of DsbA, a key bacterial oxidative folding catalyst

Since its discovery in 1991, the bacterial periplasmic oxidative folding catalyst DsbA has been the focus of intense research. Early studies addressed why it is so oxidizing and how it is maintained in its less stable oxidized state. The crystal structure of Escherichia coli DsbA (EcDsbA) revealed that the oxidizing periplasmic enzyme is a distant evolutionary cousin of the reducing cytoplasmic enzyme thioredoxin. Recent significant developments have deepened our understanding of DsbA function, mechanism, and interactions: the structure of the partner membrane protein EcDsbB, including its complex with EcDsbA, proved a landmark in the field. Studies of DsbA machineries from bacteria other than E. coli K-12 have highlighted dramatic differences from the model organism, including a striking divergence in redox parameters and surface features. Several DsbA structures have provided the first clues to its interaction with substrates, and finally, evidence for a central role of DsbA in bacterial virulence has been demonstrated in a range of organisms. Here, we review current knowledge on DsbA, a bacterial periplasmic protein that introduces disulfide bonds into diverse substrate proteins and which may one day be the target of a new class of anti-virulence drugs to treat bacterial infection. Antioxid. Redox Signal. 14, 1729–1760.

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 Å and belonged to space groups P21, P21212 and C2, respectively.

Remodelling the malignant phenotype : impact of EMT

Epithelial to mesenchymal transition (EMT) is a highly conserved programme of events essential for normal development. As it underpins cell plasticity, migration and invasion in a number of settings it is an attractive concept to apply to the malignant phenotype. Much is known about the molecular events regulating EMT in a number of model organisms, including Drosophila, sea urchin, zebrafish, xenopus and the chick. It is increasingly apparent that EMT is a highly conserved process, and that discoveries made in these systems provide excellent insights into the understanding of the role of EMT in human malignancies.

High-yeld RNA-extraction method for saliva

BACKGROUND: The use of salivary diagnostics is increasing because of its noninvasiveness, ease of sampling, and the relatively low risk of contracting infectious organisms. Saliva has been used as a biological fluid to identify and validate RNA targets in head and neck cancer patients. The goal of this study was to develop a robust, easy, and cost-effective method for isolating high yields of total RNA from saliva for downstream expression studies. METHODS: Oral whole saliva (200 mu L) was collected from healthy controls (n = 6) and from patients with head and neck cancer (n = 8). The method developed in-house used QIAzol lysis reagent (Qiagen) to extract RNA from saliva (both cell-free supernatants and cell pellets), followed by isopropyl alcohol precipitation, cDNA synthesis, and real-time PCR analyses for the genes encoding beta-actin ("housekeeping" gene) and histatin (a salivary gland-specific gene). RESULTS: The in-house QIAzol lysis reagent produced a high yield of total RNA (0.89 -7.1 mu g) from saliva (cell-free saliva and cell pellet) after DNase treatment. The ratio of the absorbance measured at 260 nm to that at 280 nm ranged from 1.6 to 1.9. The commercial kit produced a 10-fold lower RNA yield. Using our method with the QIAzol lysis reagent, we were also able to isolate RNA from archived saliva samples that had been stored without RNase inhibitors at -80 degrees C for >2 years. CONCLUSIONS: Our in-house QIAzol method is robust, is simple, provides RNA at high yields, and can be implemented to allow saliva transcriptomic studies to be translated into a clinical setting.

Saliva as an emerging biofluid for clinical diagnosis and applications of MEMS/NEMS in salivary diagnostics

Saliva as a biological fluid is gaining wider acceptance for diagnosing diseases. The growing interest in saliva as a biological fluid is due to its noninvasiveness, ease of use, cost-effectiveness, and multiple sample collection possibilities as well as minimal risk to health care professionals of contracting infectious organisms such as HIV and Hep B. However, the clinical translation of saliva is hampered by our lack of understanding of the biomolecular transportation from blood into saliva, the diurnal variations of biomolecules present in saliva, and relatively low levels of analytes (100th to a 1000th fold less than in blood). We provide information on the current status of salivary research, salivary diagnostics empowered by nanotechnology, and future prospects in this emerging field of saliva diagnostics.