Practical considerations in improving research through public involvement.

There is huge commitment to public and patient involvement (PPI) in UK clinical research. Despite there being wide agreement to practice PPI and national guidance on the subject, there are few practical examples of how to implement PPI and few published models to demonstrate how it can be achieved consistently and constructively across a broad portfolio of studies. The Peninsula Research Bank demonstrates how good PPI can be effectively integrated into experimental medicine research.

PCR-based detection of non-indigenous microorganisms in 'pristine' environments.

PCR-based technologies are widely employed for the detection of specific microorganisms, and may be applied to the identification of non-indigenous microorganisms in 'pristine' environments. For 'pristine' environments such as those found on the Antarctic continent, the application of these methods to the assessment of environmental contamination from human activities must be treated with caution. Issues such as the possibility of non-human dispersal of organisms, stability and survival of non-indigenous organisms in vivo, the sensitivity, reproducibility and specificity of the PCR process (and particularly primer design) and the sampling regime employed must all be considered in detail. We conclude that despite these limitations, PCR and related technologies offer enormous scope for assessment of both natural and non-indigenous microbial distributions.

Nanoarchaeal 16S rRNA gene sequences are widely dispersed in hyperthermophilic and mesophilic halophilic environments.

The Nanoarchaeota, proposed as the fourth sub-division of the Archaea in 2002, are known from a single isolate, Nanoarchaeum equitans, which exists in a symbiotic association with the hyperthermophilic Crenarchaeote, Ignicoccus. N. equitans fails to amplify with standard archaeal 16S PCR primers and can only be amplified using specifically designed primers. We have designed a new set of universal archaeal primers that amplify the 16S rRNA gene of all four archaeal sub-divisions, and present two new sets of Nanoarchaeota-specific primers based on all known nanoarchaeal 16S rRNA gene sequences. These primers can be used to detect N. equitans and have generated nanoarchaeal amplicons from community DNA extracted from Chinese, New Zealand, Chilean and Tibetan hydrothermal sites. Sequence analysis indicates that these environments harbour novel nanoarchaeal phylotypes, which, however, do not cluster into clear phylogeographical clades. Mesophilic hypersaline environments from Inner Mongolia and South Africa were analysed using the nanoarchaeal-specific primers and found to contain a number of nanoarchaeal phylotypes. These results suggest that nanoarchaeotes are not strictly hyperthermophilic organisms, are not restricted to hyperthermophilic hosts and may be found in a large range of environmental conditions.

Bacterial diversity in the rhizosphere of Proteaceae species.

The Cape Floral Kingdom is an area of unique plant biodiversity in South Africa with exceptional concentrations of rare and endemic species and experiencing drastic habitat loss. Here we present the first molecular study of the microbial diversity associated with the rhizosphere soil of endemic plants of the Proteaceae family (Leucospermum truncatulum and Leucadendron xanthoconus). Genomic DNA was extracted from L. truncatulum rhizosphere soil, L. xanthoconus rhizosphere and non-rhizosphere soil and used as a template for the polymerase chain reaction (PCR) amplification of the 16S ribosomal RNA gene (rDNA). Construction and sequencing of 16S rDNA libraries revealed a high level of biodiversity and led to the identification of several novel bacterial phylotypes. The bacterial community profiles were compared by 16S rDNA denaturing gradient gel electrophoresis (DGGE). Cluster analysis and biodiversity indices revealed that the rhizosphere soil samples were more similar to each other than to non-rhizosphere soil and the rhizosphere soil contained a bacterial diversity that was richer and more equitable compared with non-rhizosphere soil. A Chloroflexus and an Azospirillum genospecies were restricted to the L. xanthoconus rhizosphere soil and Stenotrophomonas genospecies was identified in all rhizosphere soil samples but was not present in the non-rhizosphere soil. Taxon-specific nested PCR and DGGE-identified differences between the Proteaceae plant rhizosphere soil with a Frankia genospecies restricted the L. truncatulum rhizosphere. Archaea-specific rDNA PCR, DGGE and DNA sequencing revealed that Crenarcheote genospecies were excluded from the plant rhizosphere soil and only present in non-rhizosphere soil.

Prototheca richardsi, a pathogen of anuran larvae, is related to a clade of protistan parasites near the animal-fungal divergence.

Prototheca richardsi is a protist of uncertain taxonomy which mediates growth inhibition in anuran larvae. Cells of P. richardsi were isolated from tadpole faeces and DNA was purified by Qiagen chromatography. Nuclear small-subunit (18S) rDNA (ssu-rDNA) was amplified by PCR using universal primers, cloned, and six clones (two from each of three separate isolates) were sequenced. All clones yielded an essentially identical sequence of 1802 nucleotides. In situ hybridization of fluorescent Prototheca-specific oligonucleotide probes, designed using the derived 18S rDNA sequence, confirmed that the sequence was indeed from P. richardsi cells and not from other components of tadpole faeces. The P. richardsi sequence was aligned with ssu-rDNA from a range of other eukaryotes, and phylogenetic analyses were carried out using several inference methods. P. richardsi consistently and stably grouped within a novel clade that contains rDNAs from an apparently heterodisperse group of parasitic micro-organisms assigned to the class Ichthyosporea. P. richardsi is evidently misplaced in the genus Prototheca, and the authors propose its inclusion in a new genus Anurofeca.