Comparative analysis of FimB and FimE recombinase activity

FimB and FimE are site-specific recombinases, part of the λ integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four ‘half sites’. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.

The cytochrome P-450 isoenzyme CYP2E1 in the biological processing of industrial chemicals : consequences for occupational and environmental medicine

The importance of the isoform CYP2E1 of the human cytochrome P-450 superfamily of enzymes for occupational and environmental medicine is derived from its unique substrate spectrum that includes a number of highly important high-production chemicals, such as aliphatic and aromatic hydrocarbons, solvents and industrial monomers (i.a. alkanes, alkenes, aromatic and halogenated hydrocarbons). Many polymorphic genes, such as CYP2E1, show considerable differences in allelic distribution between different human populations. The polymorphic nature of the human CYP2E1 gene is significant for inter-individual differences in toxicity of its substrates. Since the substrate spectrum of CYP2E1 includes many compounds of basic relevance to industrial toxicology, a rationale for metabolic interactions of different CYP2E1 substrates is provided. In-depth research into the inter-individual phenotypic differences of human CYP2E1 enzyme activities was enabled by the recognition that the 6-hydroxylation of the drug chlorzoxazone is mediated by CYP2E1. Studies on CYP2E1 phenotyping have pointed to inter-individual variations in enzyme activities. There are consistent ethnic differences in CYP2E1 enzyme expression, mostly demonstrated between European and Japanese populations, which point to a major impact of genetic factors. The most frequently studied genetic polymorphisms are the restriction fragment length polymorphisms PstI/RsaI (mutant allele: CYP2E1*5B) located in the 5′-flanking region of the gene, as well as the DraI polymorphism (mutant allele: CYP2E1*6) located in intron 6. These polymorphisms are partly related, as they form the common allele designated CYP2E1*5A. Striking inter-ethnic differences between Europeans and Asians appear with respect to the frequencies of the CYP2E1*5A allele (only approximately 5% of Europeans are heterozygous, but 37% of Asians are, whilst 6% of Asians are homozygous). Available studies indicate a wide variation in human CYP2E1 expression, which are very likely based on complex gene-environment interactions. Major inter-ethnic differences are apparent on the genotyping and the phenotyping levels. Selected cases are presented where inter-ethnic variations of CYP2E1 may provide likely explanations for unexplained findings concerning industrial chemicals that are CYP2E1 substrates. Possible consequences of differential inter-individual and inter-ethnic susceptibilities are related to individual expressions of clinical symptoms of chemical toxicity, to results of biological monitoring of exposed workers, and to the interpretation of results of epidemiological or molecular-epidemiological studies.

Markers of genetic susceptibility in human environmental hygiene and toxicology : the role of selected CYP, NAT and GST genes

Inherited genetic traits co-determine the susceptibility of an individual to a toxic chemical. Special emphasis has been put on individual responses to environmental and industrial carcinogens, but other chronic diseases are of increasing interest. Polymorphisms of relevant xenobiotic metabolising enzymes may be used as toxicological susceptibility markers. A growing number of genes encoding enzymes involved in biotransformation of toxicants and in cellular defence against toxicant-induced damage to the cells has been identified and cloned, leading to increased knowledge of allelic variants of genes and genetic defects that may result in a differential susceptibility toward environmental toxicants. "Low penetrating" polymorphisms in metabolism genes tend to be much more common in the population than allelic variants of "high penetrating" cancer genes, and are therefore of considerable importance from a public health point of view. Positive associations between cancer and CYP1A1 alleles, in particular the *2C I462V allele, were found for tissues following the aerodigestive tract. Again, in most cases, the effect of the variant CYP1A1 allele becomes apparent or clearer in connection with the GSTM1 null allele. The CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squameous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has also pointed to interactive effects. Of particular interest for the industrial and environmental field is the isozyme CYP2E1. Several genotypes of this isozyme have been characterised which seem to be associated with different levels of expression of enzyme activity. The acetylator status for NAT2 can be determined by genotyping or by phenotyping. In the pathogenesis of human bladder cancer due to occupational exposure to "classical" aromatic amines (benzidine, 4-aminodiphenyl, 1-naphthylamine) acetylation by NAT2 is regarded as a detoxication step. Interestingly, the underlying European findings of a higher susceptibility of slow acetylators towards aromatic amines are in contrast to findings in Chinese workers occupationally exposed to aromatic amines which points to different mechanisms of susceptibility between European and Chinese populations. Regarding human bladder cancer, the hypothesis has been put forward that genetic polymorphism of GSTM1 might be linked with the occurrence of this tumour type. This supports the hypothesis that exposure to PAH might causally be involved in urothelial cancers. The human polymorphic GST catalysing conjugation of halomethanes, dihalomethanes, ethylene oxide and a number of other industrial compounds could be characterised as a class theta enzyme (GSTT1) by means of molecular biology. "Conjugator" and "non-conjugator" phenotypes are coincident with the presence and absence of the GSTT1 gene. There are wide variations in the frequencies of GSTT1 deletion (GSTT1 *0/0) among different ethnicities. Human phenotyping is facilitated by the GST activity towards methyl bromide or ethylene oxide in erythrocytes which is representative of the metabolic GSTT1 competence of the entire organism. Inter-individual variations in xenobiotic metabolism capacities may be due to polymorphisms of the genes coding for the enzymes themselves or of the genes coding for the receptors or transcription factors which regulate the expression of the enzymes. Also, polymorphisms in several regions of genes may cause altered ligand affinity, transactivation activity or expression levels of the receptor subsequently influencing the expression of the downstream target genes. Studies of individual susceptibility to toxicants and gene-environment interaction are now emerging as an important component of molecular epidemiology.

Sex workers can be screened too often : a cost-effectiveness analysis in Victoria, Australia

Objectives Commercial sex is licensed in Victoria, Australia such that sex workers are required to have regular tests for sexually transmitted infections (STIs). However, the incidence and prevalence of STIs in sex workers are very low, especially since there is almost universal condom use at work. We aimed to conduct a cost-effectiveness analysis of the financial cost of the testing policy versus the health benefits of averting the transmission of HIV, syphilis, chlamydia and gonorrhoea to clients. Methods We developed a simple mathematical transmission model, informed by conservative parameter estimates from all available data, linked to a cost-effectiveness analysis. Results We estimated that under current testing rates, it costs over $A90 000 in screening costs for every chlamydia infection averted (and $A600 000 in screening costs for each quality-adjusted life year (QALY) saved) and over $A4 000 000 for every HIV infection averted ($A10 000 000 in screening costs for each QALY saved). At an assumed willingness to pay of $A50 000 per QALY gained, HIV testing should not be conducted less than approximately every 40 weeks and chlamydia testing approximately once per year; in comparison, current requirements are testing every 12 weeks for HIV and every 4 weeks for chlamydia. Conclusions Mandatory screening of female sex workers at current testing frequencies is not cost-effective for the prevention of disease in their male clients. The current testing rate required of sex workers in Victoria is excessive. Screening intervals for sex workers should be based on local STI epidemiology and not locked by legislation.

Adverse weather effects on bus ridership

This study focuses on weather effects on daily bus ridership in Brisbane, given bus’ dominance in this city. The weather pattern of Brisbane varies by season according to its sub-tropical climate characteristics. Bus is prone to inclement weather condition as it shares the road system with general traffic. Moreover, bus stops generally offer less or sometimes no protection from adverse weather. Hence, adverse weather conditions such as rain are conjectured to directly impact on daily travel behaviour patterns. There has been limited Australian research on the impact of weather on daily transit ridership. This study investigates the relationship between rainy day and daily bus ridership for the period of 2010 to 2012. Overall, rainfall affects negatively with varying impacts on different transit groups. However, this analysis confirmed a positive relationship between consecutive rainy days (rain continuing for 3 or more days). A possible explanation could be that people may switch their transport mode to bus to avoid high traffic congestion and higher accident potentiality on rainy days. Also, Brisbane’s segregated busway (BRT) corridor works favourably towards this mode choice. Our study findings enhance the fundamental understanding of traveller behaviour, particularly mode choice behaviour under adverse weather conditions.

Association study of MTHFD1 coding polymorphisms R134K and R653Q with migraine susceptibility

Objective There is evidence that folate metabolism has a role in migraine pathophysiology, particularly in the migraine with aura subtype. In this study we investigate whether two non-synonymous single nucleotide polymorphisms (SNPs), rs1950902 (C401T; R134K) and rs2236225 (G1958A; R653Q), in MTHDF1 are associated with migraine in an Australian case-control population. Background Increased plasma levels of homocysteine (HCy), one of the metabolites produced in the folate pathway, has been found to be a risk factor for migraine. There is also a genetic link, as a common polymorphism (C667T) that reduces the catalytic activity of MTHFR, the enzyme that catalyses the formation of HCy, is associated with an increase in risk of the migraine with aura (MA) subtype. MTHFD1 is a crucial multifunctional enzyme that catalyses three separate reactions of the folate pathway and therefore variants in MTHFD1 may also influence migraine susceptibility. Methods The R134K and R653Q variants in MTHFD1 were genotyped in an Australian cohort of 520 unrelated migraineurs (162 were diagnosed with migraine without aura [MO] and 358 with MA) and 520 matched controls. Data were analysed for association with migraine and for interaction with the MTHFR C667T polymorphism. Results We find no significant differences in genotype or allele frequencies for either SNP between migraineurs and controls, or when either MO or MA cases were compared to controls. In addition these MTHFD1 polymorphisms did not appear to influence the risk of MA conferred by the MTHFR 667T allele. Conclusions We find no evidence for association of the MTHFD1 R134K and R653Q polymorphisms with migraine in our Australian case-control population. However, as folate metabolism appears to be important in migraine, particularly with respect to the aura component, future studies using high throughput methods to expand the number of SNPs in folate-related genes genotyped and investigation of interactions between SNPs may be justified.

Resilience of water supply systems in meeting the challenges posed by climate change and population growth

This research project provides a scientifically robust approach for assessing the resilience of water supply systems, which are critical infrastructure, to impacts of climate change and population growth. An approach for the identification of trigger points that allows timely and appropriate management actions to be taken to avoid catastrophic system failure is an important outcome of this project. In the current absence of a formal method to evaluate the resilience of a water supply system, the approach developed in this study was based on the characterisation of resilience of a water supply system to a range of surrogate measures. Accordingly, a set of indicators are proposed to evaluate system behaviour and logistic regression analysis was used to assess system behaviour under predicted rainfall, storage and demand conditions.

Significance of drying periods on nitrate removal in experimental biofilters

Nitrogen is an important nutrient that can impact the quality of aquatic environments when present in high concentration. Even though low concentration levels of ammonium-nitrogen have been observed in laboratory studies in bioretention basins, poor removal or even the production of nitrate-nitrogen within the filter is often recorded in such studies. Ten Perspex biofilter columns of 94 mm (internal diameter) were packed with a filter layer, transition layer and a gravel layer. While the filter layer was packed to a height of 800 mm, transition and gravel layers were packed to a composite height of 220 mm and operated with simulated stormwater in the laboratory. The filter layer contained 8% organic material by weight. A free board of 350 mm provided detention storage and head to facilitate infiltration. The columns were operated with different antecedent dry days (0 d to 21 d) and constant inflow concentration at a feed rate of 100 mL/min. Samples were collected from the outflow at different time intervals, between 2 min and 150 min from the start of outflow, and were tested for nitrate-nitrogen and total organic carbon. Washoff of organic carbon from the filter layer was observed to occur for 30 min of outflow. This indicated washoff of organic carbon from the filter itself. At the same time, a very low concentration of nitrate-nitrogen was recorded at the beginning of the outflow, indicating the effective removal of nitrate-nitrogen. We conclude that the removal of nitrate-nitrogen is insignificant during the wetting phase of a rainfall event and the process of denitrification is more pronounced during the drying phase of a rainfall event. Thus intermittent wetting and drying is crucial for the removal of nitrate-nitrogen in bioretention basins.

Diverse urban plantings managed with sufficient resource availability can increase plant productivity and arthropod diversity

Buildings structures and surfaces are explicitly being used to grow plants, and these “urban plantings” are generally designed for aesthetic value. Urban plantings also have the potential to contribute significant “ecological values” by increasing urban habitat for animals such as arthropods and by increasing plant productivity. In this study, we evaluated how the provision of these additional ecological values is affected by plant species richness; the availability of essential resources for plants, such as water, light, space; and soil characteristics. We sampled 33 plantings located on the exterior of three buildings in the urban center of Brisbane, Australia (subtropical climatic region) over 2, 6 week sampling periods characterized by different temperature and rainfall conditions. Plant cover was estimated as a surrogate for productivity as destructive sampling of biomass was not possible. We measured weekly light levels (photosynthetically active radiation), plant CO2 assimilation, soil CO2 efflux, and arthropod diversity. Differences in plant cover were best explained by a three-way interaction of plant species richness, management water regime and sampling period. As the richness of plant species increased in a planter, productivity and total arthropod richness also increased significantly—likely due to greater habitat heterogeneity and quality. Overall we found urban plantings can provide additional ecological values if essential resources are maintained within a planter such as water, light and soil temperature. Diverse urban plantings that are managed with these principles in mind can contribute to the attraction of diverse arthropod communities, and lead to increased plant productivity within a dense urban context.

Assessing rod, cone and melanopsin contributions to human pupil flicker responses

Purpose: To determine the relative contributions of rods, cones and melanopsin to pupil responses in humans using temporal sinusoidal stimulation for light levels spanning the low mesopic to photopic range. Methods: A four-primary Ganzfeld photostimulator controlled flicker stimulations at seven light levels (-2.7 to 2 log cd/m2) and five frequencies (0.5 to 8Hz). Pupil diameter was measured using a high-resolution eyetracker. Three kinds of sinusoidal photoreceptor modulations were generated using silent substitution: 1) rod modulation, 2) cone modulation, and 3) combined rod and cone modulation in phase (Experiment 1) or phase shifted (Experiment 2) from a fixed rod phase. The melanopsin excitation was computed for each condition. A vector sum model was used to estimate the relative contribution of rods, cones and melanopsin to the pupil response. Results: From Experiment 1, the pupil frequency response peaked at 1Hz at two mesopic light levels for the three modulation conditions. Analyzing the rod-cone phase difference for the combined modulations (Experiment 2) identified a V-shaped response amplitude with a minimum between 135° and 180°. The pupil response phases increased as cone modulation phase increased. The pupil amplitude increased with increasing light level for cone and combined in-phase rod and cone modulation, but not for the rod modulation. Conclusions: These results demonstrate that cone- and rod-pathway contributions are more predominant than melanopsin contribution to the phasic pupil response. The combined rod, cone and melanopsin inputs to the phasic state of the pupil light reflex follow linear summation.

Development of a transient, high-level expression platform for protein production in plants

Plants are an attractive alternative to conventional expression systems for the production of recombinant proteins and useful biologics, however, the economic viability of plant made proteins is strongly yield dependent. This study aimed to improve transgene expression levels in the plant host Nicotiana benthamiana using the Agroinfiltration transient expression platform. Independent investigation of the physical, chemical and genetic features associated with Agroinfiltration identified factors that improved transformation frequencies, elevated transgene expression levels and ultimately improved protein yield. The major outcome of this research was a novel hyper-expression system for biofarming recombinant proteins in plants.

Comparison of ocular modulation transfer function determined by a ray-tracing aberrometer and a double-pass system in early cataract patients

BACKGROUND: The evaluation of retinal image quality in cataract eyes has gained importance and the clinical modulation transfer functions (MTF) can obtained by aberrometer and double pass (DP) system. This study aimed to compare MTF derived from a ray tracing aberrometer and a DP system in early cataractous and normal eyes. METHODS: There were 128 subjects with 61 control eyes and 67 eyes with early cataract defined according to the Lens Opacities Classification System III. A laser ray-tracing wavefront aberrometer (iTrace) and a double pass (DP) system (OQAS) assessed ocular MTF for 6.0 mm pupil diameters following dilation. Areas under the MTF (AUMTF) and their correlations were analyzed. Stepwise multiple regression analysis assessed factors affecting the differences between iTrace- and OQAS-derived AUMTF for the early cataract group. RESULTS: For both early cataract and control groups, iTrace-derived MTFs were higher than OQAS-derived MTFs across a range of spatial frequencies (P < 0.01). No significant difference between the two groups occurred for iTrace-derived AUMTF, but the early cataract group had significantly smaller OQAS-derived AUMTF than did the control group (P < 0.01). AUMTF determined from both the techniques demonstrated significant correlations with nuclear opacities, higher-order aberrations (HOAs), visual acuity, and contrast sensitivity functions, while the OQAS-derived AUMTF also demonstrated significant correlations with age and cortical opacity grade. The factors significantly affecting the difference between iTrace and OQAS AUMTF were root-mean-squared HOAs (standardized beta coefficient = -0.63, P < 0.01) and age (standardized beta coefficient = 0.26, P < 0.01). CONCLUSIONS: MTFs determined from a iTrace and a DP system (OQAS) differ significantly in early cataractous and normal subjects. Correlations with visual performance were higher for the DP system. OQAS-derived MTF may be useful as an indicator of visual performance in early cataract eyes.

Performance characterisation of a stormwater treatment bioretention basin

Treatment performance of bioretention basins closely depends on hydrologic and hydraulic factors such as rainfall characteristics and inflow and outflow discharges. An in-depth understanding of the influence of these factors on water quality treatment performance can provide important guidance for effective bioretention basin design. In this paper, hydraulic and hydrologic factors impacting pollutant removal by a bioretention basin were assessed under field conditions. Outcomes of the study confirmed that the antecedent dry period plays an important role in influencing treatment performance. A relatively long antecedent dry period reduces nitrite and ammonium concentrations while increasing the nitrate concentration, which confirms that nitrification occurs within the bioretention basin. Additionally, pollutant leaching influences bioretention basin treatment performance, reducing the nutrients removal efficiency, which was lower for high rainfall events. These outcomes will contribute to a greater understanding of the treatment performance of bioretention basins, assisting in the design, operation and maintenance of these systems.

Inner-ear morphology of the New Zealand Kiwi (Apteryx mantelli) suggests high-frequency specialization

The sensory systems of the New Zealand kiwi appear to be uniquely adapted to occupy a nocturnal ground-dwelling niche. In addition to well-developed tactile and olfactory systems, the auditory system shows specializations of the ear, which are maintained along the central nervous system. Here, we provide a detailed description of the auditory nerve, hair cells, and stereovillar bundle orientation of the hair cells in the North Island brown kiwi. The auditory nerve of the kiwi contained about 8,000 fibers. Using the number of hair cells and innervating nerve fibers to calculate a ratio of average innervation density showed that the afferent innervation ratio in kiwi was denser than in most other birds examined. The average diameters of cochlear afferent axons in kiwi showed the typical gradient across the tonotopic axis. The kiwi basilar papilla showed a clear differentiation of tall and short hair cells. The proportion of short hair cells was higher than in the emu and likely reflects a bias towards higher frequencies represented on the kiwi basilar papilla. The orientation of the stereovillar bundles in the kiwi basilar papilla showed a pattern similar to that in most other birds but was most similar to that of the emu. Overall, many features of the auditory nerve, hair cells, and stereovilli bundle orientation in the kiwi are typical of most birds examined. Some features of the kiwi auditory system do, however, support a high-frequency specialization, specifically the innervation density and generally small size of hair-cell somata, whereas others showed the presumed ancestral condition similar to that found in the emu.

Recent surveys of bats (Mammalia: Chiroptera) from China. I. Rhinolophidae and Hipposideridae

We conducted surveys of bats in China between 1999 and 2007, resulting in the identification of at least 62 species. In this paper we present data on 19 species, comprising 12 species from the family Rhinolophidae and seven from the Hipposideridae. Rhinolophids captured were Rhinolophus affinis, R. ferrumequinum, R. lepidus, R. luctus, R. macrotis, R. siamensis, R. marshalli, R. rex, R. pearsonii, R. pusillus, R. sinicus and R. stheno. Because of extensive morphological similarities we question the species distinctiveness of R. osgoodi (may be conspecific with R. lepidus), R. paradoxolophus (which may best be treated as a subspecies of R. rex), R. huananus (probably synonymous with R. siamensis), and we are skeptical as to whether R. sinicus is distinct from R. thomasi. Hipposiderids captured were Hipposideros armiger, H. cineraceus, H. larvatus, H. pomona, H. pratti, Aselliscus stoliczkanus and Coelops frithii. Of these species, two rhinolophids (Rhinolophus marshalli and R. stheno) and one hipposiderid (Hipposideros cineraceus) represent new species records for China. We present data on species' ranges, morphology and echolocation call frequencies, as well as some notes on ecology and conservation status. China hosts a considerable diversity of rhinolophid and hipposiderid bats, yet threats to their habitats and populations are substantial.