Marked deterioration in rheumatoid arthritis associated bronchiectasis following treatment with Rituximab.

We report a 67 year old lady with Rheumatoid Arthritis (RA) and mild bronchiectasis (BE) whose treatment was escalated to Rituximab. Nine months after commencing Rituximab her lung sepsis worsened dramatically with repeated hospitalization, new sputum isolation of Stenotrophomonas maltophilia and Pseudomonas aeruginosa and marked radiological deterioration in BE. She was found to have a low serum IgG and IgM levels almost certainly as a complication of Rituximab. Immunoglobulin replacement therapy was instituted and her clinical status has slowly improved.

Microbial DNA profiling by multiplex terminal restriction fragment length polymorphism for forensic comparison of soil and the influence of sample condition.

AIMS: To evaluate: (i) the impact of air-drying on bacterial, archaeal and fungal soil DNA profiles and (ii) the potential use of multiplex-terminal restriction fragment length polymorphism (M-TRFLP) as a tool for forensic comparison of soil. METHODS AND RESULTS: An M-TRFLP approach was used to profile bacterial, archaeal and fungal DNA profiles from five different soil sites. Air-drying soil significantly reduced the quantity of DNA but the number of operational taxanomic units (OTU) was unaffected. The impact of air-drying on soil DNA profiles was dependent on soil site and microbial primers. Fungal profiles were altered the least by air-drying. For prokaryotic profiles, air-drying altered the relative similarity/dissimilarity between soil sites. The M-TRFLP approach was more discriminatory compared with soil colour and single-taxa profiling, but did not significantly improve resolution between two similar soils. CONCLUSIONS: Of those tested, soil fungi were potentially the more robust target for application to soil forensic studies as they were altered less by air-drying and provided clear discrimination of soils from different sites. The M-TRFLP method demonstrated potential to achieve greater resolution, discriminating the soil sites based on both bacterial and fungal components. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil DNA profiling has potential as a forensic tool, but sample condition and the appropriate selection of microbial target taxa must be considered.

FragMatch--a program for the analysis of DNA fragment data.

FragMatch is a user-friendly Java-supported program that automates the identification of taxa present in mixed samples by comparing community DNA fragment data against a database of reference patterns for known species. The program has a user-friendly Windows interface and was primarily designed for the analysis of fragment data derived from terminal restriction fragment length polymorphism analysis of ectomycorrhizal fungal communities, but may be adapted for other applications such as microsatellite analyses. The program uses a simple algorithm to check for the presence of reference fragments within sample files that can be directly imported, and the results appear in a clear summary table that also details the parameters that were used for the analysis. This program is significantly more flexible than earlier programs designed for matching RFLP patterns as it allows default or user-defined parameters to be used in the analysis and has an unlimited database size in terms of both the number of reference species/individuals and the number of diagnostic fragments per database entry. Although the program has been developed with mycorrhizal fungi in mind, it can be used to analyse any DNA fragment data regardless of biological origin. FragMatch, along with a full description and users guide, is freely available to download from the Aberdeen Mycorrhiza Group web page (http://www.aberdeenmycorrhizas.com).

Linking biosensor responses to Cd, Cu and Zn partitioning in soils.

Soils bind heavy metals according to fundamental physico-chemical parameters. Bioassays, using bacterial biosensors, were performed in pore waters extracted from 19 contrasting soils individually amended with Cd, Cu and Zn concentrations related to the EU Sewage Sludge Directive. The biosensors were responsive to pore waters extracted from Zn amended soils but less so to those of Cu and showed no toxicity to pore water Cd at these environmentally relevant amended concentrations. Across the range of soils, the solid-solution heavy metal partitioning coefficient (K(d)) decreased (p<0.01) with increasing amendments of Cu and Zn; Cu exhibited the highest K(d) values. Gompertz functions of Cu and Zn, K(d) values against luminescence explained the relationship between heavy metals and biosensors. Consequently, biosensors provide a link between biologically defined hazard assessments of metals and standard soil-metal physico-chemical parameters for determining critical metal loadings in soils.

Microbial indicators of heavy metal contamination in urban and rural soils.

Urban soils and especially their microbiology have been a neglected area of study. In this paper, we report on microbial properties of urban soils compared to rural soils of similar lithogenic origin in the vicinity of Aberdeen city. Significant differences in basal respiration rates, microbial biomass and ecophysiological parameters were found in urban soils compared to rural soils. Analysis of community level physiological profiles (CLPP) of micro-organisms showed they consumed C sources faster in urban soils to maintain the same level activity as those in rural soils. Cu, Pb, Zn and Ni were the principal elements that had accumulated in urban soils compared with their rural counterparts with Pb being the most significant metal to distinguish urban soils from rural soils. Sequential extraction showed the final residue after extraction was normally the highest proportion except for Pb, for which the hydroxylamine-hydrochloride extractable Pb was the largest part. Acetic acid extractable fraction of Cd, Cu, Ni, Pb and Zn were higher in urban soils and aqua regia extractable fraction were lower suggesting an elevated availability of heavy metals in urban soils. Correlation analyses between different microbial indicators (basal respiration, biomass-C, and sole C source tests) and heavy metal fractions indicated that basal respiration was negatively correlated with soil Cd, Cu, Ni and Zn inputs while soil microbial biomass was only significantly correlated with Pb. However, both exchangeable and iron- and manganese-bound Ni fractions were mostly responsible for shift of the soil microbial community level physiological profiles (sole C source tests). These data suggest soil microbial indicators can be useful indicators of pollutant heavy metal stress on the health of urban soils.

Bacterial diversity promotes community stability and functional resilience after perturbation.

The relationships between bacterial community diversity and stability were investigated by perturbing soils, with naturally differing levels of diversity, to equivalent toxicity using copper sulfate and benzene. Benzene amendment led to large decreases in total bacterial numbers and biomass in both soils. Benzene amendment of an organo-mineral/improved pasture soil altered total soil bacterial community structure but, unlike amendment of the mineral/arable soil, maintained genetic diversity, based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis targeting DNA and RNA, until week 9 of the perturbation experiment. Assuming equivalent toxicity, the genetic diversity of the naturally more diverse soil was more resistant to benzene perturbation than the less diverse soil. The broad scale function (mineralization of 14C-labelled wheat shoot) of both benzene- and copper-treated soil communities was unaffected. However, narrow niche function (mineralization of 14C-labelled 2,4-dichlorophenol) was impaired for both benzene-polluted soils. The organo-mineral soil recovered this function by the end of the experiment but the mineral soil did not, suggesting greater resilience in the more diverse soil. Despite a large reduction in bacterial numbers and biomass in the copper-treated soils, only small differences in bacterial community diversity were observed by week 9 in the copper-polluted soils. The overall community structure was little altered and functionality, measured by mineralization rates, remained unchanged. This suggested a non-selective pressure and a degree of genetic and functional resistance to copper perturbation, despite a significant reduction in bacterial numbers and biomass. However, initial shifts in physiological profiles of both copper-polluted soils were observed but rapidly returned to those of the controls. This apparent functional recovery, accompanied by an increase in culturability, possibly reflects adaptation by the surviving communities to perturbation. The findings indicate that, although soil communities may be robust, relationships between diversity and stability need to be considered in developing a predictive understanding of response to environmental perturbations.

Pine microsatellite markers allow roots and ectomycorrhizas to be linked to individual trees.

Linking roots and ectomycorrhizas (EcM) to individual host trees in the field is required to test whether individual trees support different ectomycorrhizal communities. Here we describe a method that identifies the source of EcM roots by PCR of polymorphic pine nuclear microsatellite loci using fluorescently labelled primers and high-throughput fragment analysis. ITS-PCR can also be performed on the same EcM DNA extract for fungal identification. The method was tested on five neighbouring Scots pine (Pinus sylvestris var scotica) trees in native woodland. Successful host tree identification from DNA extracts of EcM root tips was achieved for 93% of all root fragments recovered from soil cores. It was estimated that each individual mature pine sampled was colonised by between 15 and 19 EcM fungi. The most abundant fungal species were found on all five trees, and within the constraints of the sampling scheme, no differences between trees in EcM fungal community structure or composition were detected.

Two newborns with nutritional vitamin B12 deficiency: challenges in newborn screening for vitamin B12 deficiency.

Vitamin B12 deficiency causes decreased Methionine Synthase and L-Methylmalonyl-CoA Mutase activity and results in accumulation of Homocysteine, Methylmalonic acid and Propionylcarnitine. Propionylcarnitine is included in tandem mass spectrometry-based newborn screening programs for detection of certain inborn errors of metabolism. We report two asymptomatic newborns with Vitamin B12 deficiency due to maternal deficiencies. One was detected incidentally at 3 weeks of age; the second on supplemental newborn screening based on elevated Propionylcarnitine at 2 days of age. This illustrates the potential for false negative results for Vitamin B12 deficiency screening by acylcarnitine profiling in newborn screening. Homocysteine and Methylmalonic acid may be better markers of Vitamin B12 deficiency. In conclusion, we suggest measuring Methylmalonic acid, Propionylcarnitine and Homocysteine levels in blood spots in expanded newborn screening in order to detect asymptomatic newborns with Vitamin B12 deficiency. Further studies are needed to establish the sensitivity of these three markers in screening for Vitamin B12 deficiency.

Methods for rearing Syngaster lepidus and Jarra phoracantha (Hymenoptera: Braconidae), larval parasitoids of the phloem-colonizing longhorned beetles Phoracantha semipunctata and P. recurva (Coleoptera: Cerambycidae).

Two species of cerambycid beetles that attack eucalypts, Phoracantha semipunctata (Fabricius) and P. recurva Newman, have been accidentally introduced from Australia into most regions of the world in which their hosts have been planted. The beetles cause extensive mortality in plantations and landscape plantings of the trees. Management programmes have focused on integration of silvicultural practices, host plant resistance and biological control. To rear and release natural enemies of the larval stages of the beetles in California, mass rearing protocols for continuous production of two species of parasitoids have been developed. The methods described represent the first long-term and large-scale techniques for mass rearing parasitoids of any wood-boring cerambycid species. In addition to providing large numbers of parasitoids for releases, the mass rearing effort has also provided large numbers of parasitoids for fundamental studies of their biology and behaviour.

Microbial communities in different soil types do not converge after diesel contamination.

AIMS: To study the comparative effect of diesel addition and simulated bioremediation on the microbial community in three different soil types. METHODS AND RESULTS: Three different soils were amended with diesel and bioremediation treatment simulated by addition of nutrients. The progress of bioremediation, and the effect on the indigenous microbial communities, was monitored using microbiological techniques. These included basal respiration, sole carbon source utilization patterns using both a commercially-available substrate set and a set designed to highlight changes in hydrocarbon-utilizing bacteria, and phospholipid fatty acid (PLFA) profiling. The development of active hydrocarbon-degrading communities was indicated by the disappearance of diesel, increases in soil respiration and biomass, and large changes in the sole carbon source utilization patterns and PLFA profiles compared with control soils. However, comparison of the relative community structure of the three soils using PLFA profiling showed that there was no tendency for the community structure of the three different soil types to converge as a result of contamination. In fact, they became more dissimilar as a result. Changes in the sole carbon source utilization patterns using the commercially-available set of carbon sources indicated the same result as shown by PLFA profiling. The specially selected set of carbon sources yielded no additional information compared with the commercially-available set. CONCLUSIONS: Diesel contamination does not result in the development of similar community profiles in different soil types. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that different soils have different inherent microbial potential to degrade hydrocarbons, a finding that should be taken into account in impact and risk assessments. Following the development of the microbial community and its recovery is a useful and sensitive way of monitoring the impact and recovery of oil-contaminated soils.

An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material.

Arbuscular mycorrhizal fungi (order Glomales), which form mycorrhizal symbioses with two out of three of all plant species, are believed to be obligate biotrophs that are wholly dependent on the plant partner for their carbon supply. It is thought that they possess no degradative capability and that they are unable to decompose complex organic molecules, the form in which most soil nutrients occur. Earlier suggestions that they could exist saprotrophically were based on observation of hyphal proliferation on organic materials. In contrast, other mycorrhizal types have been shown to acquire nitrogen directly from organic sources. Here we show that the arbuscular mycorrhizal symbiosis can both enhance decomposition of and increase nitrogen capture from complex organic material (grass leaves) in soil. Hyphal growth of the fungal partner was increased in the presence of the organic material, independently of the host plant.

Comparison of response of six different luminescent bacterial bioassays to bioremediation of five contrasting oils.

The performance of six different bioluminescent bacteria for the assessment of oil bioremediation was compared. Three contained lux genes linked to promoters from hydrocarbon degradation pathways: Pseudomonas fluorescens HK44 (pUTK21), Escherichia coli HMS174 (pOS25) and E. coli DH5 alpha (pGEc74, pJAMA7), responding to naphthalene, isopropylbenzene and octane, respectively. The other three expressed lux constitutively: E. coli HB101 (pUCD607) and P. putida F1 (pUCD607) are genetically engineered, while Vibrio fischeri is naturally bioluminescent and was included to facilitate comparison with previous work. Five different oils (four crude oils plus diesel) were spiked into soil, and the progress of remediation was followed over a period of 119 d by monitoring both hydrocarbon disappearance and changes in the microbial response to soil extracts. The octane bioassay was the only one of the hydrocarbon-responsive bacterial assays to show any appreciable response, with up to 20-fold induction by light crude oils. Heavy crude oil and diesel elicited a much weaker response. The metabolic (lux constitutively expressed) bioassays showed that there was a general increase in toxicity over the course of the experiment, although toxicity to E. coli HB101 (pUCD607) appeared to be decreasing by the final sampling point. The metabolic bioassay response was much less variable between the different oils than for the first three, catabolic, strains.

The effect of culture conditions on the mycelial growth and luminescence of naturally bioluminescent fungi.

The effects of temperature, light and pH on mycelial growth and luminescence of four naturally bioluminescent fungi were investigated. Cultures of Armillaria mellea, Mycena citricolor, Omphalotus olearius and Panellus stipticus were grown at 5 degrees C, 15 degrees C, 22 degrees C and 30 degrees C, under 24 h light, 12 h light/12 h dark and 24 h dark, and at a pH ranging from 3.5 to 7 in three separate experiments. Temperature and pH had a significant effect on mycelial growth and bioluminescence, however light did not. Bioluminescence and mycelial growth were optimum at 22 degrees C and pH 3-3.5, the exception being M. citricolor for which bioluminescence and growth were optimum at pH 5-6 and pH 4, respectively. With the exception of M. citricolor, bioluminescence and mycelial growth were greater under 24 h darkness. An understanding of the effect of culture conditions on mycelial growth and luminescence is necessary for the future application of bioluminescent fungi as biosensors.

Development of QSARs to investigate the bacterial toxicity and biotransformation potential of aromatic heterocylic compounds.

A series of aromatic heterocyclic and hydrocarbon compounds were tested for toxicity and biotransformation potential against two contrasting lux-marked whole-cell microbial biosensors. Toxicity was determined by inhibition of light output of a Pseudomonas fluorescens construct that expresses lux constitutively. Biotransformation was tested by increase in light output of P. fluorescens HK44 (pUTK21), which expresses lux when in the presence of a metabolic intermediate (salicylate). The data were then modelled against physical/chemical properties of the compounds tested to see if quantitative structure-activity relationships (QSARs) could be derived. Toxicity was found to be accurately predicted by log Kow (R2 = 0.95, Q2 = 0.88), with the basic (pyridine-ring containing) heterocycles modelled separately. The biotransformation data were best modelled using lowest unoccupied molecular orbital (LUMO) energies (R2 = 0.90, Q2 = 0.87).

The effect of EDTA and fulvic acid on Cd, Zn, and Cu toxicity to a bioluminescent construct (pUCD607) of Escherichia coli.

The hypothesis, that metal toxicity is dominated by free ion activity, was tested by comparing calculated metal activities with measured toxic responses to a genetically modified, luminescent bacterium, Escherichia coli. The toxicity of Cd, Cu, and Zn sulphate salts in the presence of EDTA and fulvic acid in well-defined solutions was measured. Good agreement between free metal activity and toxicity was found for Cu but not for Zn and Cd. The toxicity relationships were altered by glucose addition to the organism. Stable chloride complexes may have contributed to the toxicity of Cd under the test conditions. The results suggest that there is not always a simple relationship between toxicity and free-ion metal concentration and that further account should be taken of competitive interactions between living cells and ligands and the physiological status of the organism.

Investigating the specificity of regulators of degradation of hydrocarbons and hydrocarbon-based compounds using structure-activity relationships.

Microbial biosensors which have genes for bioluminescence coupled to genes that control hydrocarbon degradation pathways can be used as reporters on the specificity of regulation of those pathways. Structure-activity relationships can be used to discover what governs that specificity, and can also be used to separate compounds into different groups depending on mode of action. Published data for four different bioluminescent biosensors, reporting on toluene (two separate biosensors), isopropylbenzene, and octane, were analyzed to develop structure-activity relationships between biological response and physical/chemical properties. Good QSARs (quantitative structure-activity relationships) were developed for three out of the four biosensors, with between 88 and 100 per cent of the variance explained. Parameters found to be important in controlling regulator specificity were hydrophobicity, lowest unoccupied molecular orbital energies, and molar volume. For one of the biosensors, it was possible to show that the biological response to chemicals tested fell into three separate classes (non-hydrocarbons, aliphatic hydrocarbons, and aromatic hydrocarbons). A statistically significant QSAR based on hydrophobicity was developed for the fourth biosensor, but was poor in comparison to the other three (44 per cent variance explained).

Characterisation of rhizobia from African acacias and other tropical woody legumes using Biolog and partial 16S rRNA sequencing.

A Biolog (sole carbon source utilisation) user database of tropical and temperature rhizobial strains was created and used in conjunction with the partial 16S rRNA sequencing method to characterise 12 rhizobial isolates from African acacias and other tropical woody legumes. There was close agreement between the two methods but also some significant discrepancies. A high degree of diversity was shown in the relatively small sample of isolates, with 4 out of 5 of the currently proposed rhizobial genera represented. This is the first time Biolog has shown congruence with genotypic fingerprinting using a wide selection of rhizobial reference and test strains.

The Central Asylum for the Instruction of the Deaf and Dumb, Canajoharie, New York, 1823-1835.

The Central Asylum for the Instruction of the Deaf and Dumb, Canajoharie, NY, was a public school for the Deaf that existed from 1823 to 1835. Little has been written about it. This study draws upon as much information as appears to be available on the history of this school. The history of the Central Asylum adds to the understanding of the beginnings of public education for the Deaf in the United States.